Stabilized human immunodeficiency virus (hiv) envelope (env) trimer vaccines and methods of using same

ABSTRACT

The invention features stabilized human immunodeficiency virus (HIV) envelope (Env) trimers. The invention also features vaccines, nucleic acids, and vectors to deliver and/or facilitate production of the stabilized HIV Env trimers. In addition, the invention features methods of making and using the stabilized HIV Env trimers of the invention.

STATEMENT AS TO FEDERALLY FUNDED RESEARCH

This invention was made with government support under Grant Nos.A1096040 and A1084794, awarded by the National Institutes of Health(NIH)/National Institute of Allergy and Infectious Diseases (NIAID). Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

Vaccines that elicit cellular immune responses against viruses seek toreflect global viral diversity in order to effectively treat or preventviral infection. For HIV vaccines, the initiation of robust and diversehuman immunodeficiency virus (HIV)-specific T cell responses isdesirable for an effective HIV vaccine. The highly variable Envelopeprotein (Env) is the primary target for neutralizing antibodies againstHIV, and vaccine antigens may be tailored accordingly to elicit theseantibody responses. To this end, immunogens mimicking the trimericstructure of Env on the native HIV virion are actively being pursued asantibody-based HIV vaccines. However, it has proven difficult to producebiochemically stable trimeric Env immunogens that elicit diverseneutralizing antibody responses.

Thus, there is an unmet need in the field for the development ofvaccines that include novel, optimized trimeric Env immunogens, whichcan elicit broadly neutralizing antibody responses in order to allow formore successful HIV vaccination outcomes.

SUMMARY OF THE INVENTION

In a first aspect, the invention features a stabilized trimer havingthree gp140 polypeptides in which at least one (e.g., two or each) ofthe gp140 polypeptides includes an amino acid sequence having at least90% identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity) to, or the sequence of, SEQ ID NO: 2 (mEnv+).

In a related second aspect, the invention features a stabilized trimerhaving three gp140 polypeptides in which at least one (e.g., two oreach) of the gp140 polypeptides includes an amino acid sequence havingsubstantially the sequence of (e.g., 99% or more identity), or thesequence of, SEQ ID NO: 1 (mEnv) or SEQ ID NO: 3 (cEnv).

In some embodiments, the stabilized trimers are heterotrimers. Thestabilized polypeptide heterotrimers may include two mosaic Env1 gp140polypeptides (e.g., mEnv and/or mEnv+) each including an amino acidsequence having at least 90% identity (e.g., at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identity) to, or the sequence of, SEQ IDNO: 1 or 2, and one clade C Env gp140 polypeptide (e.g., “cEnv” havingSEQ ID NO: 3) including an amino acid sequence having at least 90%identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity) to, or the sequence of, SEQ ID NO: 3 (cEnv) (e.g., two mEnvand one cEnv; two mEnv+ and one cEnv; or one mEnv, one mEnv+, and onecEnv). In other embodiments, the stabilized heterotrimers may includeone mosaic Env1 gp140 polypeptide (e.g., mEnv and/or mEnv+) including anamino acid sequence having at least 90% identity (e.g., at least 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to, or the sequenceof, SEQ ID NO: 1 or 2, and two clade C Env gp140 polypeptides (e.g.,cEnv) each including an amino acid sequence having at least 90% (e.g.,at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to, orthe sequence of, SEQ ID NO: 3 (e.g., one mEnv and two cEnv; one mEnv+and two cEnv). In some embodiments, the stabilized heterotrimer includesa combination of two different mosaic Env1 sequences (e.g., one mEnv andtwo mEnv+; two mEnv and one mEnv+; or one mEnv, one mEnv+, and cEnv). Insome embodiments, the stabilized heterotrimer includes cEnv and two ofthe same Env1 polypeptides (e.g., two mEnv and one cEnv; two mEnv+ andone cEnv). In other embodiments, the stabilized heterotrimer includesone cEnv and two different mosaic Env1 polypeptides (e.g., one cEnv, onemEnv, and one mEnv+). In yet other embodiments, the stabilizedheterotrimer includes two cEnv polypeptides and one mosaic Env1polypeptide (e.g., two cEnv and one mEnv; or two cEnv and one mEnv+).

Alternatively, stabilized gp140 Env trimers can be prepared in which oneor two of the gp140 Env polypeptides in the trimer has a sequence of SEQID NO: 4 (mosaic gp140 Env2, “mEnv2”) or SEQ ID NO: 5 (mosaic gp140Env3, “mEnv3”). In another embodiment said stabilized trimers have threegp140 polypeptides in which at least one (e.g., two or each) of thegp140 polypeptides includes an amino acid sequence having at least 90%identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity) to, or the sequence of, SEQ ID NO:4 or 5. Preferably, mEnv2 ormEnv3 is modified in a similar manner to that of mEnv, mEnv+, or cEnv,which each possess a trimerization domain, as discussed herein below.Therefore, in some embodiments of the invention, stabilized gp140 Envtrimers can be prepared which have the following constituentpolypeptides: one mEnv and two mEnv2; two mEnv and one mEnv2; one mEnv+and two mEnv2; two mEnv+ and one mEnv2; one cEnv and two mEnv2; two cEnvand one mEnv2; one mEnv, one mEnv+, and one mEnv2; one mEnv, one cEnv,and mEnv2; one mEnv+, one cEnv, and one mEnv2; one mEnv and two mEnv3;two mEnv and one mEnv3; one mEnv+ and two mEnv3; two mEnv+ and onemEnv3; one cEnv and two mEnv3; two cEnv and one mEnv3; one mEnv, onemEnv+, and one mEnv3; one mEnv, one cEnv, and mEnv3; one mEnv+, onecEnv, and one mEnv3; one mEnv, one mEnv2, and one mEnv3; one mEnv+, onemEnv2, and one mEnv3; or one cEnv, one mEnv2, and one mEnv3.

In a third aspect, the invention features a composition including astabilized trimer of the first or second aspect. In an embodiment, thecomposition of the third aspect includes one or more differentstabilized trimer(s). In other embodiments, the different stabilizedtrimer(s) has three gp140 polypeptides in which at least one (e.g., twoor each) of the gp140 polypeptides comprises an amino acid sequencehaving at least 90% identity (e.g., at least 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity) to, or the sequence of, SEQ ID NOs: 1,2, or 3. In other embodiments, the different stabilized trimer(s) may bea homotrimer or a heterotrimer. In some embodiments, the compositions ofthe third aspect further include a pharmaceutically acceptable carrier,excipient, or diluent, and/or an adjuvant.

In a fourth aspect, the invention features a vaccine including any oneof the compositions of the third aspect. In some embodiments, thevaccine is used for treating or reducing the risk of a humanimmunodeficiency virus (HIV) infection in a subject in need thereof. Insome embodiments, the vaccine elicits production of neutralizinganti-HIV antisera (e.g., neutralizing anti-HIV-1 antisera) afteradministration to the subject. The anti-HIV antisera can neutralize HIV(e.g., HIV-1), for example, selected from any one or more of clade A,clade B, and clade C.

In a fifth aspect, the invention features a nucleic acid molecule havinga nucleotide sequence that encodes at least one (e.g., two, or three ormore) gp140 polypeptide, wherein the at least one gp140 polypeptideincludes: (a) an amino acid sequence having at least 95% identity (e.g.,at least 96%, 97%, 98%, or 99% identity) to, or the sequence of, SEQ IDNO: 1; (b) an amino acid sequence having at least 95% identity (e.g., atleast 96%, 97%, 98%, or 99% identity) to, or the sequence of, SEQ ID NO:2; or (c) an amino acid sequence having the sequence of SEQ ID NO: 3;(d) an amino acid sequence having the sequence of SEQ ID NO:4; (e) anamino acid sequence having the sequence of SEQ ID NO:5 or combinationsthereof. In some embodiments, the nucleic acid molecule further includesa nucleotide sequence that encodes one or more different (e.g., asecond, third, or fourth) gp140 polypeptides (e.g., gp140 polypeptideshaving at least 95% identity (e.g., at least 96%, 97%, 98%, or 99%identity) to, or the sequence of, SEQ ID NO: 1, 2, and/or 3). In someembodiments, the nucleic acid molecule includes one or more internalribosome entry site (IRES) sequences to allow for the expression ofmultiple peptide or polypeptide chains from the single nucleic acidmolecule transcript.

In a sixth aspect, the invention features a vector including one or morenucleic acid molecules of the fifth aspect. In some embodiments, thevector is an adenovirus vector or a poxvirus vector. The adenovirusvector may be derived, for example, from a recombinant adenovirusserotype 11 (Ad11), adenovirus serotype 15 (Ad15), adenovirus serotype24 (Ad24), adenovirus serotype 26 (Ad26), adenovirus serotype 34 (Ad34),adenovirus serotype 35 (Ad35), adenovirus serotype 48 (Ad48), adenovirusserotype 49 (Ad49), adenovirus serotype 50 (Ad50), Pan9 (AdC68), or achimeric variant thereof (e.g., adenovirus serotype 5 HVR48 (Ad5HVR48)).The poxvirus vector may be derived, for example, from modified vacciniavirus Ankara (MVA).

In a seventh aspect, the invention provides a method of treating orreducing the risk of an HIV infection in a subject in need thereof byadministering a therapeutically effective amount of a composition of theinvention (e.g., any one of the stabilized trimers of the first orsecond aspect, the compositions of the third aspect, the vaccines of thefourth aspect, the nucleic acid molecules of the fifth aspect, and/orthe vectors of the sixth aspect) to the subject, such as a mammal, forexample, a human. Treating, according to this seventh aspect of theinvention, can be therapeutic or prophylactic.

In an eighth aspect, the invention provides a method of reducing anHIV-mediated activity in a subject infected with HIV by administering atherapeutically effective amount of a composition of the invention(e.g., any one of the stabilized trimers of the first or second aspect,the compositions of the third aspect, the vaccines of the fourth aspect,the nucleic acid molecules of the fifth aspect, and/or the vectors ofthe sixth aspect) to the subject. In some embodiments, the HIV-mediatedactivity is viral spread, infection, or cell fusion. Cell fusion may be,for example, target cell entry or syncytial formation. In someembodiments, the HIV titer in the subject infected with HIV is decreased(e.g., by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more compared to HIV titer of the subject prior totreatment or a control subject infected with HIV but not treated withthe composition(s) of the invention) after administration of the vaccineto the subject, such as a mammal, for example, a human.

In some embodiments, the composition (e.g., a vaccine) is administeredintramuscularly, intravenously, intradermally, percutaneously,intraarterially, intraperitoneally, intralesionally, intracranially,intraarticularly, intraprostatically, intrapleurally, intratracheally,intranasally, intravitreally, intravaginally, intrarectally, topically,intratumorally, peritoneally, subcutaneously, subconjunctivally,intravesicularlly, mucosally, intrapericardially, intraumbilically,intraocularly, orally, topically, locally, by inhalation, by injection,by infusion, by continuous infusion, by localized perfusion bathingtarget cells directly, by catheter, by lavage, by gavage, in cremes, orin lipid compositions. In some embodiments, the subject is administeredat least one dose (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more doses)of the composition or is administered at least one dose (e.g., 1, 2, 3,4, 5, 6, 7, 8, 9, 10, or more doses) daily, weekly, monthly, or yearly.The administration period may be defined (e.g., 1-4 weeks, 1-12 months,1-20 years) or may be for the life of the subject. In other embodiments,the subject is administered at least two doses (e.g., 2, 3, 4, 5, 6, 7,8, 9, 10, or more doses) of the composition. In yet another embodiment,the composition is administered to said subject as a prime or a boostcomposition or in a prime-boost regimen. In a preferred embodiment, oneor more composition(s) (e.g., a vaccine) of the invention isadministered as a boost.

In another preferred embodiment, the invention features a method oftreating or reducing the risk of an HIV infection in a subject byadministering, as the prime composition in a prime-boost vaccinationregimen, a vaccine that includes a first polypeptide having at least 85%amino acid sequence identity (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to, or the sequence of,SEQ ID NO: 6, or at least a first vector (e.g., an adenoviral orpoxvirus vector) that includes a first nucleic acid molecule thatencodes this first polypeptide. Optionally, a second polypeptide havingat least 85% identity (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identity) to, or the sequence of, SEQ IDNO: 7 may also be administered in combination with the firstpolypeptide, or, if a first vector encoding the first polypeptide isadministered, a second vector (e.g., an adenoviral or poxvirus vector)including a second nucleic acid molecule that encodes the secondpolypeptide may be administered in combination with the first vector.The boost composition in this prime-boost regimen may include one ormore of the composition(s) of the invention (e.g., any one of thestabilized trimers of the first or second aspect, the compositions ofthe third aspect, the vaccines of the fourth aspect, the nucleic acidmolecules of the fifth aspect, and/or the vectors of the sixth aspect).In still other embodiments, the prime composition in this prime-boostregimen may include polypeptide(s) having the sequence(s) of any one ofSEQ ID NOs: 8-32, or one or more vectors including nucleic acidmolecules that encode any one of SEQ ID NOs: 8-32, followed by a boostincluding one or more of the composition(s) of the invention (e.g., anyone of the stabilized trimers of the first or second aspect, thecompositions of the third aspect, the vaccines of the fourth aspect, thenucleic acid molecules of the fifth aspect, and/or the vectors of thesixth aspect).

In still other embodiments, one or more composition(s) of the invention(e.g., a vaccine) is administered as the prime composition in aprime-boost regimen and the boost composition is a different vaccinecomposition, e.g., a vaccine that includes one or more polypeptideshaving at least 85% amino acid sequence identity (e.g., 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to,or the sequence of, any one or more of SEQ ID NOs: 6-32 (preferably thepolypeptide of SEQ ID NO: 6 and/or 7), or one or more vectors (e.g.,adenoviral or poxvirus vectors) each of which includes a nucleic acidmolecule that encodes one or more polypeptides having at least 85%identity (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identity) to, or the sequence of, one or more of SEQ IDNOs: 8-32 (preferably the vector encodes the polypeptide of SEQ ID NO: 6and/or 7).

In some embodiments, the subject may, for example, be administeredpolypeptide compositions of the invention (e.g., stabilized gp140 Envtrimers of the invention) in a non-vectored composition. The polypeptidecomposition administered may include between approximately 1 μg and 1 mgof stabilized Env trimers, and more preferably between 50 μg and 300 μgof stabilized Env trimers of the invention.

In other embodiments wherein the delivery vector is a virus, the subjectcan be administered at least about 1×10³ viral particles (vp)/dose orbetween 1×10¹ and 1×10¹⁴ vp/dose, preferably between 1×10³ and 1×10¹²vp/dose, and more preferably between 1×10⁵ and 1×10¹¹ vp/dose. Thecomposition may be administered, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 30, 35, 40, 45, 50, 55, or 60 minutes, 2, 4, 6, 10, 15, or24 hours, 2, 3, 5, or 7 days, 2, 4, 6 or 8 weeks, or even 3, 4, or 6months pre-exposure or pre-diagnosis, or may be administered to thesubject 15-30 minutes or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 20, 24, 48,or 72 hours, 2, 3, 5, or 7 days, 2, 4, 6 or 8 weeks, 3, 4, 6, or 9months, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 years or longerpost-diagnosis or post-exposure or to HIV. The subject is administeredone or more doses of the composition once daily, weekly, monthly, oryearly. When treating an HIV infection, the composition(s) of theinvention (e.g., any one of the stabilized trimers of the first, second,or third aspect, the compositions of the fourth or fifth aspect, thevaccines of the sixth aspect, the nucleic acid molecules of the seventhaspect, and/or the vectors of the eighth aspect) may be administered tothe subject either before the occurrence of symptoms of an HIV infectionor disease/syndrome (e.g., acquired immune deficiency syndrome (AIDS))or a definitive diagnosis, or after diagnosis or symptoms becomeevident. The composition(s) may be administered, for example,immediately after diagnosis or the clinical recognition of symptoms or2, 4, 6, 10, 15, or 24 hours, 2, 3, 5, or 7 days, 2, 4, 6 or 8 weeks, oreven 3, 4, or 6 months after diagnosis or detection of symptoms.

In a ninth aspect, the invention provides methods of manufacturing avaccine for treating or reducing the risk of an HIV infection in asubject in need thereof. The method includes the steps of: (a)contacting a nucleic acid of the second aspect of the invention (e.g., anucleic acid that further includes a vector of the eighth aspect) with acell; and (b) expressing the nucleic acid in the cell to form astabilized trimer. In some embodiments, the method is performed in vitroor ex vivo. In some embodiments, the cell is a bacterial, plant, ormammalian cell (e.g., a human or non-human mammalian cell). In apreferred embodiment, the mammalian cell is a 293T cell.

In a final aspect, the invention features a kit including: (a) acomposition of the invention (e.g., any one of the stabilized trimers ofthe first, second, or third aspect, the compositions of the fourth orfifth aspect, the vaccines of the sixth aspect, the nucleic acidmolecules of the seventh aspect, and/or the vectors of the eighthaspect, e.g., a vaccine including mEnv and/or mEnv+ trimers and cEnvtrimers); (b) a pharmaceutically acceptable carrier, excipient, ordiluent; and (c) instructions for use thereof. The kit may optionallyinclude an adjuvant.

In preferred embodiments of all aspects of the invention, the subject isa mammal, preferably a primate, such as a human.

Definitions

As used herein, the term “about” means +/−10% of the recited value.

By “adenovirus” is meant a medium-sized (90-100 nm), non-envelopedicosahedral virus that includes a capsid and a double-stranded linearDNA genome. The adenovirus can be a naturally occurring, but isolated,adenovirus (e.g., sAd4287, sAd4310A, or sAd4312) or a recombinantadenovirus (e.g., replication-defective or replication competentsAd4287, sAd4310A, or sAd4312, or a chimeric variant thereof).

As used herein, “administering” is meant a method of giving a dosage ofa pharmaceutical composition (e.g., a composition of the invention, suchas any one of the vaccines of the first or fourth aspects, thecompositions of the third aspect, the nucleic acid molecules of thefifth aspect, and/or the vectors of the sixth aspect) to a subject. Thecompositions utilized in the methods described herein can beadministered, for example, intramuscularly, intravenously,intradermally, percutaneously, intraarterially, intraperitoneally,intralesionally, intracranially, intraarticularly, intraprostatically,intrapleurally, intratracheally, intranasally, intravitreally,intravaginally, intrarectally, topically, intratumorally, peritoneally,subcutaneously, subconjunctivally, intravesicularlly, mucosally,intrapericardially, intraumbilically, intraocularly, orally, topically,locally, by inhalation, by injection, by infusion, by continuousinfusion, by localized perfusion bathing target cells directly, bycatheter, by lavage, by gavage, in cremes, or in lipid compositions. Thepreferred method of administration can vary depending on various factors(e.g., the components of the composition being administered and theseverity of the condition being treated).

As used herein, the term “clade” refers to related humanimmunodeficiency viruses (HIVs) classified according to their degree ofgenetic similarity. There are currently three groups of HIV-1 isolates:M, N and O. Group M (major strains) consists of at least ten clades, Athrough J. Group O (outer strains) may consist of a similar number ofclades. Group N is a new HIV-1 isolate that has not been categorized ineither group M or O. In certain exemplary embodiments, a composition ofthe invention (e.g., any one of the vaccines of the first or fourthaspects, the compositions of the third aspect, the nucleic acidmolecules of the fifth aspect, and/or the vectors of the sixth aspect)as described herein will recognize and raise an immune response (e.g.,neutralizing anti-HIV antisera) against two, three, four, five, six,seven, eight, nine, ten or more clades and/or two or more groups of HIV.

Throughout this specification and claims, the word “comprise,” orvariations such as “comprises” or “comprising,” will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

As used herein, the term “envelope glycoprotein” refers, but is notlimited to, the glycoprotein that is expressed on the surface of theenvelope of HIV virions and the surface of the plasma membrane of HIVinfected cells. The env gene encodes gp160, which is proteolyticallycleaved into the gp120 and gp41 Envelope (Env) proteins. Gp120 binds tothe CD4 receptor on a target cell that has such a receptor, such as,e.g., a T-helper cell. Gp41 is non-covalently bound to gp120, andprovides the second step by which HIV enters the cell. It is originallyburied within the viral envelope, but when gp120 binds to a CD4receptor, gp120 changes its conformation causing gp41 to become exposed,where it can assist in fusion with the host cell.

By “gene product” is meant to include mRNAs transcribed from a gene aswell as polypeptides translated from those mRNAs.

By “heterologous nucleic acid molecule” or “heterologous gene” is meantany exogenous nucleic acid molecule (e.g., a nucleic acid moleculeencoding an optimized gp140 Env polypeptide of the invention) that canbe inserted into the a vector of the invention (e.g., an adenovirus orpoxvirus vector) for transfer into a cell, tissue, or organism, forsubsequent expression of a gene product of interest or fragment thereofencoded by the heterologous nucleic acid molecule or gene. In apreferred embodiment, the heterologous nucleic acid molecule, which canbe administered to a cell or subject as part of the present invention,can include, but is not limited to, a nucleic acid molecule encoding atleast one optimized mosaic Env polypeptide (e.g., a mosaic Env1polypeptide, such as mEnv and mEnv+) and/or a clade C Env polypeptide(e.g., a clade C Env1 polypeptide, such as cEnv).

By “human immunodeficiency virus” or “HIV” is meant a virus of the genusLentivirinae, part of the family of Retroviridae, and includes, but isnot limited to, HIV type 1 (HIV-1) and HIV type 2 (HIV-2), two speciesof HIV that infect humans.

By “immune response” is meant any response to an antigen or antigenicdeterminant by the immune system of a subject (e.g., a human). Exemplaryimmune responses include humoral immune responses (e.g., production ofantigen-specific antibodies, e.g., neutralizing antibodies (NAbs)) andcell-mediated immune responses (e.g., lymphocyte proliferation).

As used herein, the term “reducing” with respect to HIV refers to areduction or decrease of an HIV-mediated activity (e.g., infection,fusion (e.g., target cell entry and/or syncytia formation), viralspread, etc.) and/or a decrease in viral titer. HIV-mediated activityand/or HIV titer may be decreased by 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or morecompared to that of a control subject (e.g., an untreated subject or asubject treated with a placebo).

By “neutralizing antibody” or “NAb” is meant an antibody which either ispurified from, or is present in, serum and which recognizes a specificantigen (e.g., HIV Env glycoprotein, such as a gp140 polypeptide or agp120 polypeptide) and inhibits the effect(s) of the antigen in the host(e.g., a human). As used herein, the antibody can be a single antibodyor a plurality of antibodies.

“Nucleic acid” or “polynucleotide,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase, or by a syntheticreaction. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and their analogs. If present, modification tothe nucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter synthesis, such as by conjugation with a label. Other types ofmodifications include, for example, “caps,” substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, poly-L-lysine, etc.), those with intercalators (e.g.,acridine, psoralen, etc.), those containing chelators (e.g., metals,radioactive metals, boron, oxidative metals, etc.), those containingalkylators, those with modified linkages (e.g., alpha anomeric nucleicacids, etc.), as well as unmodified forms of the polynucleotide(s).Further, any of the hydroxyl groups ordinarily present in the sugars maybe replaced, for example, by phosphonate groups, phosphate groups,protected by standard protecting groups, or activated to prepareadditional linkages to additional nucleotides, or may be conjugated tosolid or semi-solid supports. The 5′ and 3′ terminal OH can bephosphorylated or substituted with amines or organic capping groupmoieties of from 1 to 20 carbon atoms. Other hydroxyls may also bederivatized to standard protecting groups. Polynucleotides can alsocontain analogous forms of ribose or deoxyribose sugars that aregenerally known in the art, including, for example, 2′-O-methyl-,2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclic sugar analogs,alpha-anomeric sugars, epimeric sugars such as arabinose, xyloses orlyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclicanalogs and a basic nucleoside analogs such as methyl riboside. One ormore phosphodiester linkages may be replaced by alternative linkinggroups. These alternative linking groups include, but are not limitedto, embodiments wherein phosphate is replaced by P(O)S (“thioate”),P(S)S (“dithioate”), “(O)NR₂ (“amidate”), P(O)R, P(O)OR′, CO or CH₂(“formacetal”), in which each R or R′ is independently H or substitutedor unsubstituted alkyl (1-20 C) optionally containing an ether (—O—)linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not alllinkages in a polynucleotide need be identical. The precedingdescription applies to all polynucleotides referred to herein, includingRNA and DNA.

By “optimized” is meant an immunogenic polypeptide that is not anaturally-occurring peptide, polypeptide, or protein, such as anon-naturally occurring viral polypeptide (e.g., a gp140 polypeptide ofthe invention). Optimized viral polypeptide sequences are initiallygenerated by modifying the amino acid sequence of one or morenaturally-occurring viral gene products (e.g., peptides, polypeptides,and proteins, e.g., a viral Env polypeptide, e.g., a viral Env1, Env2,and/or Env3 polypeptide) to increase the breadth, intensity, depth, orlongevity of the antiviral immune response (e.g., cellular or humoralimmune responses) generated upon immunization (e.g., when incorporatedinto a composition of the invention, e.g., vaccine of the invention) ofa subject (e.g., a human). Thus, the optimized viral polypeptide maycorrespond to a “parent” viral gene sequence; alternatively, theoptimized viral polypeptide may not correspond to a specific “parent”viral gene sequence but may correspond to analogous sequences fromvarious strains or quasi-species of a virus. Modifications to the viralgene sequence that can be included in an optimized viral polypeptideinclude amino acid additions, substitutions, and deletions. In oneembodiment of the invention, the optimized polypeptide is a mosaicenvelope protein, such as mosaic Env1 gp140 (see, e.g., U.S. PatentPublication No. 2012/0076812, herein incorporated by reference), or anoptimized version thereof, which has been further altered to include aleader/signal sequence for maximal protein expression, cleavage sitemutation(s), a factor Xa site, and/or a foldon trimerization domain(see, e.g., SEQ ID NO: 2). Methods of generating an optimized viralpolypeptide are described in, e.g., Fisher et al. “Polyvalent Vaccinefor Optimal Coverage of Potential T-Cell Epitopes in Global HIV-1Variants,” Nat. Med. 13(1):100-106 (2007) and International PatentApplication Publication WO 2007/024941, herein incorporated byreference. Once the optimized viral polypeptide sequence is generated,the corresponding polypeptide can be produced or administered bystandard techniques (e.g., recombinant viral vectors, such as theadenoviral vectors disclosed in International Patent ApplicationPublications WO 2006/040330 and WO 2007/104792, herein incorporated byreference) and optionally assembled in conjunction with one or moreother viral polypeptides of the invention to form a stabilizedpolypeptide trimer.

By “pharmaceutically acceptable diluent, excipient, carrier, oradjuvant” is meant a diluent, excipient, carrier, or adjuvant which isphysiologically acceptable to the subject while retaining thetherapeutic properties of the pharmaceutical composition with which itis administered. One exemplary pharmaceutically acceptable carrier isphysiological saline. Other physiologically acceptable diluents,excipients, carriers, or adjuvants and their formulations are known toone skilled in the art (see, e.g., U.S. Pub. No. 2012/0076812).

By “recombinant,” with respect to a composition (e.g., a vector of theinvention, such as an adenovirus or poxvirus vector), is meant acomposition that has been manipulated in vitro (e.g., using standardcloning techniques) to introduce changes (e.g., changes to thecomposition, e.g., adenovirus or poxvirus genome of an adenovirus orpoxvirus vector, respectively) that enable binding to or containment ofa therapeutic agent and/or that promote the introduction of atherapeutic agent into a subject (e.g., a human) or a host cell. Therecombinant composition of the invention may therefore be an adenoviralor poxviral transport vector (e.g., a replication-defective adenoviralor poxviral vector) for delivery of one or more of the stabilized Envpolypeptide trimers of the invention.

By “sequence identity” or “sequence similarity” is meant that theidentity or similarity between two or more amino acid sequences, or twoor more nucleotide sequences, is expressed in terms of the identity orsimilarity between the sequences. Sequence identity can be measured interms of “percentage (%) identity,” wherein the higher the percentage,the more identity shared between the sequences. Sequence similarity canbe measured in terms of percentage similarity (which takes into accountconservative amino acid substitutions); the higher the percentage, themore similarity shared between the sequences. Homologs or orthologs ofnucleic acid or amino acid sequences possess a relatively high degree ofsequence identity/similarity when aligned using standard methods.Sequence identity may be measured using sequence analysis software onthe default setting (e.g., Sequence Analysis Software Package of theGenetics Computer Group, University of Wisconsin Biotechnology Center,1710 University Avenue, Madison, Wis. 53705). Such software may matchsimilar sequences by assigning degrees of homology to varioussubstitutions, deletions, and other modifications.

As used herein, the term “stabilized polypeptide trimer” refers, but isnot limited to, an oligomer that includes a protein and/or polypeptidesequence that increases the stability (e.g., via the presence of one ormore oligomerization domains) of the trimeric structure (e.g., reducesdissociation of a trimer into monomeric units). In particular, thestabilized polypeptide trimer is composed of three mosaic Env proteins(e.g., Env1, Env2, and/or Env3), three clade C Env proteins, or acombination of one or more mosaic Env proteins and one or more clade CEnv proteins, in which at least one Env protein includes anoligomerization domain. An “oligomerization domain” refers, but is notlimited to, a polypeptide sequence that can be used to increase thestability of an oligomeric envelope protein such as, e.g., to increasethe stability of a HIV gp140 trimer. Oligomerization domains can be usedto increase the stability of homooligomeric polypeptides as well asheterooligomeric polypeptides. Oligomerization domains are well known inthe art, and include “trimerization domains.” A trimerization domainrefers to an oligomerization domain that stabilizes trimericpolypeptides (e.g., trimers consisting of one or more of the gp140polypeptides of the invention). Examples of trimerization domainsinclude, but are not limited to, the T4-fibritin “foldon” trimerizationdomain; the coiled-coil trimerization domain derived from GCN4 (Yang etal. (2002) J. Virol. 76:4634); and the catalytic subunit of E. coliaspartate transcarbamoylase as a trimer tag (Chen et al. (2004) J.Virol. 78:4508).

A “subject” is a vertebrate, such as a mammal (e.g., a human). Mammalsalso include, but are not limited to, farm animals (such as cows), sportanimals (e.g., horses), pets (such as cats and dogs), mice, and rats. Asubject to be treated according to the methods described herein (e.g., asubject having an HIV infection or a subject at risk of an HIVinfection) may be one who has been diagnosed by a medical practitioneras having such a condition. Diagnosis may be performed by any suitablemeans. A subject in whom the risk of an HIV infection is to be reducedor prevented may or may not have received such a diagnosis. One skilledin the art will understand that a subject to be treated according to thepresent invention may have been subjected to standard tests or may havebeen identified, without examination, as one at high risk due to thepresence of one or more risk factors (e.g., a needle stick or knownexposure to HIV or an HIV infected individual).

By “having substantially the sequence of” with respect to constructs ofthe invention is meant having at least 99% sequence identity to arecited reference sequence (e.g., having no more than 7 amino acidresidue differences, e.g., 1, 2, 3, 4, 5, or 6 amino acid residuedifferences (e.g., additions, deletions, or conservative amino acidsubstitutions), relative to a recited reference sequence).

By “therapeutically effective amount” is meant an amount of atherapeutic agent that alone, or together with one or more additional(optional) therapeutic agents, produces beneficial or desired resultsupon administration to a mammal. The therapeutically effective amountdepends upon the context in which the therapeutic agent is applied. Forexample, in the context of administering a vaccine composition includinga therapeutic agent such as a stabilized gp140 trimer of the invention,the therapeutically effective amount of the vaccine composition is anamount sufficient to achieve a reduction in the level of HIV (e.g., asmeasured by a stabilization or decrease in HIV titer compared to anon-treated control), and/or an increase in the level of neutralizinganti-HIV antisera (e.g., as measured by an increase in serumneutralizing antibody levels relative to a non-treated control in aluciferase-based virus neutralization assay) as compared to a responseobtained without administration of a composition of the invention (e.g.,a vaccine composition), and/or to prevent the propagation of aninfectious virus (e.g., HIV) in a subject (e.g., a human) having anincreased risk of viral infection. Ideally, a therapeutically effectiveamount provides a therapeutic effect without causing a substantialcytotoxic effect in the subject. In general, a therapeutically effectiveamount of a composition administered to a subject (e.g., a human) willvary depending upon a number of factors associated with that subject,for example the overall health of the subject, the condition to betreated, or the severity of the condition. A therapeutically effectiveamount of a composition can be determined by varying the dosage of theproduct and measuring the resulting therapeutic response.

As used herein, and as well understood in the art, “treatment” is anapproach for obtaining beneficial or desired results, such as clinicalresults. Beneficial or desired results can include, but are not limitedto, alleviation or amelioration of one or more symptoms or conditions;diminishment of extent of disease, disorder, or condition; stabilization(i.e., not worsening) of a state of disease, disorder, or condition;prevention of spread of disease, disorder, or condition; delay orslowing the progress of the disease, disorder, or condition;amelioration or palliation of the disease, disorder, or condition; andremission (whether partial or total), whether detectable orundetectable. “Palliating” a disease, disorder, or condition means thatthe extent and/or undesirable clinical manifestations of the disease,disorder, or condition are lessened and/or time course of theprogression is slowed or lengthened, as compared to the extent or timecourse in the absence of treatment.

The term “vaccine,” as used herein, is defined as material used toprovoke an immune response (e.g., the production of neutralizinganti-HIV antisera). Administration of the vaccine to a subject mayconfer at least partial immunity against HIV infection.

As used herein, the term “vector” is meant to include, but is notlimited to, a virus (e.g., adenovirus or poxvirus), naked DNA,oligonucleotide, cationic lipid (e.g., liposome), cationic polymer(e.g., polysome), virosome, nanoparticle, or dentrimer. By “adenovirusvector” is meant a composition that includes one or more genes(non-structural or structural), or fragments thereof, from an adenoviralspecies (e.g., adenovirus serotype 11 (Ad11), adenovirus serotype 15(Ad15), adenovirus serotype 24 (Ad24), adenovirus serotype 26 (Ad26),adenovirus serotype 34 (Ad34), adenovirus serotype 35 (Ad35), adenovirusserotype 48 (Ad48), adenovirus serotype 49 (Ad49), adenovirus serotype50 (Ad50), Pan9 (AdC68), or a chimeric variant thereof (e.g., adenovirusserotype 5 HVR48 (Ad5HVR48))) that may be used to transmit one or moreheterologous genes (e.g., one or more of the optimized gp140polypeptides of the invention) from a viral or non-viral source to asubject or a host. The nucleic acid material of the viral vector may beencapsulated, e.g., in a lipid membrane or by structural proteins (e.g.,capsid proteins), that may include one or more viral polypeptides (e.g.,an envelope glycoprotein). The viral vector can be used to infect cellsof a subject, which, in turn, promotes the translation of theheterologous gene(s) of the viral vector into a protein product (e.g.,one or more of the gp140 Env polypeptides described herein, such that astabilized trimer of the invention is formed).

The term “virus,” as used herein, is defined as an infectious agent thatis unable to grow or reproduce outside a host cell and that infectsmammals (e.g., humans) or birds.

Other features and advantages of the invention will be apparent from thefollowing Detailed Description, the drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the amino acid sequence of a mosaic human immunodeficiencyvirus (HIV) gp140 Envelope (Env) polypeptide (mEnv; SEQ ID NO: 1) of theinvention. The boxed region identifies the signal/leader sequence; theunderlined region identifies gp120; the plain text region identifies thegp41 ectodomain; and the double-underscored region identifies theT4-fibritin “foldon” trimerization/oligomerization domain.

FIG. 1B shows the amino acid sequence of a mosaic HIV gp140 Envpolypeptide (mEnv+; SEQ ID NO: 2) of the invention. This polypeptidesequence has been further optimized and includes a differentsignal/leader sequence to maximize protein expression (boxed region);the addition of cleavage site-inactivating mutations (E/E substitutionmutations) (circled residues); and the addition of a Factor Xa site(zig-zag underlined region). Other regions are noted as in FIG. 1A.

FIG. 1C shows the amino acid sequence of an optimized clade C Envpolypeptide (cEnv; SEQ ID NO: 3) of the invention. All regions are notedas in FIG. 1B.

FIG. 2 is a Western blot showing the expression levels of mEnv and mEnv+in lanes 3 and 4, respectively, compared to cEnv and an expressionvector control (pVRC8400) in lanes 1 and 2, respectively.

FIG. 3 is a gel filtration chromatograph depicting the uniform elutionof mEnv+ trimers six days post-PEI transfection of 293T cells in rollerbottles (750-ml of supernatant).

FIG. 4 is an image of a 4-16% gradient SDS-PAGE showing the peakfractions of mEnv+ following gel filtration elution. The final proteinyield per purification is approximately 8.44 mg following gelfiltration. The final concentration is approximately 5.62 mg/ml.

FIG. 5A is a graph showing a quantitative analysis of ID₅₀ titermeasuring TZM.b1 neutralizing antibody responses in guinea pigspre-vaccination (Pre) and post-vaccination (Post) with clade C gp140 Env(cEnv) homotrimer tested against a multi-clade panel of tier 1neutralization-sensitive isolates including clade B (SF162.LS andBa1.26) and clade C (MW965.26 and TV1.21) HIV-1 Envelope pseudoviruses,as well as Murine leukemia virus (MuLV) (negative control).

FIG. 5B is a graph showing a quantitative analysis of ID₅₀ titermeasuring TZM.b1 neutralizing antibody responses in guinea pigspre-vaccination (Pre) and post-vaccination (Post) with mosaic gp140 Envversion-1 (mEnv) homotrimer tested against a multi-clade panel of tier 1neutralization-sensitive isolates including clade B (SF162.LS andBa1.26) and clade C (MW965.26 and TV1.21) HIV-1 Envelope pseudoviruses,as well as Murine leukemia virus (MuLV) (negative control).

FIG. 5C is a graph showing a quantitative analysis of ID₅₀ titermeasuring TZM.b1 neutralizing antibody responses in guinea pigspre-vaccination (Pre) and post-vaccination (Post) with both cEnv andmEnv trimers tested against a multi-clade panel of tier 1neutralization-sensitive isolates including clade B (SF162.LS andBa1.26) and clade C (MW965.26 and TV1.21) HIV-1 Envelope pseudoviruses,as well as Murine leukemia virus (MuLV) (negative control).

FIG. 6A is a graph showing a quantitative analysis of ID₅₀ titermeasuring TZM.b1 neutralizing antibody responses in guinea pigspre-vaccination (Pre) and post-vaccination (Post) with cEnv homotrimer,mEnv homotrimer, or both cEnv and mEnv trimers tested against a Tier 1Bintermediate neutralization-sensitive clade A HIV-1 Envelopepseudovirus, MS208.A1.

FIG. 6B is a graph showing a quantitative analysis of ID₅₀ titermeasuring TZM.b1 neutralizing antibody responses in guinea pigspre-vaccination (Pre) and post-vaccination (Post) with cEnv homotrimer,mEnv homotrimer, or both cEnv and mEnv trimers tested against a Tier 1Bintermediate neutralization-sensitive clade A HIV-1 Envelopepseudovirus, Q23.17.

FIG. 7A is a graph showing a quantitative analysis of ID₅₀ titermeasuring TZM.b1 neutralizing antibody responses in guinea pigspre-vaccination (Pre) and post-vaccination (Post) with cEnv homotrimer,mEnv homotrimer, or both cEnv and mEnv trimers tested against a Tier 1Ahighly neutralization-sensitive clade B HIV-1 Envelope pseudovirus,SF162.LS.

FIG. 7B is a graph showing a quantitative analysis of ID₅₀ titermeasuring TZM.b1 neutralizing antibody responses in guinea pigspre-vaccination (Pre) and post-vaccination (Post) with cEnv homotrimer,mEnv homotrimer, or both cEnv and mEnv trimers tested against a Tier 1Bintermediate neutralization-sensitive clade B HIV-1 Envelopepseudovirus, BaL.26.

FIG. 7C is a graph showing a quantitative analysis of ID₅₀ titermeasuring TZM.b1 neutralizing antibody responses in guinea pigspre-vaccination (Pre) and post-vaccination (Post) with cEnv homotrimer,mEnv homotrimer, or both cEnv and mEnv trimers tested against a Tier 1Bintermediate neutralization-sensitive clade B HIV-1 Envelopepseudovirus, SS1196.1.

FIG. 7D is a graph showing a quantitative analysis of ID₅₀ titermeasuring TZM.b1 neutralizing antibody responses in guinea pigspre-vaccination (Pre) and post-vaccination (Post) with cEnv homotrimer,mEnv homotrimer, or both cEnv and mEnv trimers tested against a Tier 1Bintermediate neutralization-sensitive clade B HIV-1 Envelopepseudovirus, 6535.3.

FIG. 8A is a graph showing a quantitative analysis of ID₅₀ titermeasuring TZM.b1 neutralizing antibody responses in guinea pigspre-vaccination (Pre) and post-vaccination (Post) with cEnv homotrimer,mEnv homotrimer, or both cEnv and mEnv trimers tested against a Tier 1Ahighly neutralization-sensitive clade C HIV-1 Envelope pseudovirus,MW965.26.

FIG. 8B is a graph showing a quantitative analysis of ID₅₀ titermeasuring TZM.b1 neutralizing antibody responses in guinea pigspre-vaccination (Pre) and post-vaccination (Post) with cEnv homotrimer,mEnv homotrimer, or both cEnv and mEnv trimers tested against a Tier 1Bintermediate neutralization-sensitive clade C HIV-1 Envelopepseudovirus, TV1.21.

FIG. 8C is a graph showing a quantitative analysis of ID₅₀ titermeasuring TZM.b1 neutralizing antibody responses in guinea pigspre-vaccination (Pre) and post-vaccination (Post) with cEnv homotrimer,mEnv homotrimer, or both cEnv and mEnv trimers tested against a Tier 1Bintermediate neutralization-sensitive clade C HIV-1 Envelopepseudovirus, ZM109F.PB4.

FIG. 8D is a graph showing a quantitative analysis of ID₅₀ titermeasuring TZM.b1 neutralizing antibody responses in guinea pigspre-vaccination (Pre) and post-vaccination (Post) cEnv homotrimer, mEnvhomotrimer, or both cEnv and mEnv trimers tested against a Tier 1Bintermediate neutralization-sensitive clade C HIV-1 Envelopepseudovirus, ZM197M.PB7.

DETAILED DESCRIPTION OF THE INVENTION

Most antibodies induced by human immunodeficiency virus (HIV) (e.g., HIVtype 1 (HIV-1)) are ineffective at preventing initiation or spread ofinfection, as they are either non-neutralizing or narrowlyisolate-specific. One of the biggest challenges in HIV vaccinedevelopment is to design a HIV envelope immunogen that can induceprotective, neutralizing antibodies effective against the diverse HIVstrains that characterize the global pandemic. Indeed, the generation of“broadly neutralizing” antibodies that recognize relatively conservedregions on the envelope glycoprotein are rare. The present invention isbased in part on the discovery of stabilized trimeric HIV envelope (Env)proteins and combinations thereof that elicit a surprisingly broadneutralizing antibody response in vivo.

Stabilized Gp140 Env Trimers of the Invention

The invention features novel stabilized HIV gp140 Env polypeptidetrimers. Stabilized trimers of the invention feature optimized gp140 Envpolypeptides. These polypeptides may have, or may be modified toinclude, one or more of the following domains and/or mutations. Thegp140 Env polypeptide constituents may include a T4-fibritin “foldon”trimerization domain sequence to support stable trimer formation (see,e.g., FIGS. 1A, 1B, and 1C, depicting the amino acid sequences of mEnv(SEQ ID NO: 1), mEnv+(SEQ ID NO: 2), and cEnv (SEQ ID NO: 3),respectively, which each include a C-terminal trimerization domain). Theoptimized gp140 Env polypeptides may also include cleavage sitemutations to enhance stability, for example, by eliminating cleavage bya peptidase (see, e.g., FIGS. 1B and 1C, which depict the mutatedresidues as circled residues in the mEnv+ and cEnv amino acid sequence,respectively, between the gp120 and gp41 moieties). The optimized gp140Env polypeptides may additionally have a signal/leader sequence tomaximize protein expression (see, e.g., the signal/leader sequence ofmEnv+ or cEnv, demarcated in FIGS. 1B and 1C, respectively). Further,the optimized gp140 Env polypeptides may include a Factor Xa cleavagesite (SRIEGR), which may, for example, be incorporated upstream of(N-terminal to) the trimerization domain (see, e.g., FIGS. 1B and 1C,which depict the location of the Factor Xa cleavage site in the aminoacid sequence of mEnv+ and cEnv, respectively). As discussed hereinbelow, the stabilized trimers of the invention are preferablyhomotrimers (e.g., trimers composed of three identical polypeptides).Heterotrimers (e.g., trimers composed of three polypeptides that are notall identical) of the invention are also envisioned.

The stabilized trimers of the invention are preferably stabilizedhomotrimers that include, for example, three gp140 polypeptides, whereineach of the gp140 polypeptides includes an amino acid sequence having atleast 90% identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% identity) to, or the sequence of, SEQ ID NO: 2 (mEnv+). Theinvention also features stabilized homotrimers including three gp140polypeptides, wherein each of said gp140 polypeptides includes an aminoacid sequence having substantially the sequence of (e.g., 99% or moreidentity), or the sequence of, SEQ ID NO: 1 (mEnv) or SEQ ID NO: 3(cEnv) or SEQ ID NO:4 or SEQ ID NO:5. Exemplary homotrimers of theinvention include Trimers 1, 2, and 3 in Table 1 below.

Alternatively, the stabilized trimer of the invention may be astabilized heterotrimer. For example, the stabilized trimer may be astabilized heterotrimer that includes a combination of two differentmosaic Env1 sequences (e.g., one mEnv and two mEnv+; two mEnv and onemEnv+; or one mEnv, one mEnv+, and cEnv). In some instances, thestabilized heterotrimer includes cEnv and two of the same Env1polypeptides (e.g., two mEnv and one cEnv; two mEnv+ and one cEnv). Inother instances, the stabilized heterotrimer includes one cEnv and twodifferent mosaic Env1 polypeptides (e.g., one cEnv, one mEnv, and onemEnv+).

Alternatively, the stabilized heterotrimer may include one or twoconstituent Env polypeptides including an amino acid sequence of SEQ IDNO: 4 (mosaic gp140 Env2, “mEnv2”) or SEQ ID NO: 5 (mosaic gp140 Env3,“mEnv3”). Preferably, mEnv2 or mEnv3 is modified in a similar manner tothat of mEnv, mEnv+, or cEnv, which each possess a trimerization domain,as discussed above and as depicted in FIGS. 1A-1C. Therefore, otherstabilized heterotrimers of the invention include trimers having thefollowing constituent polypeptides: one mEnv and two mEnv2; two mEnv andone mEnv2; one mEnv+ and two mEnv2; two mEnv+ and one mEnv2; one cEnvand two mEnv2; two cEnv and one mEnv2; one mEnv, one mEnv+, and onemEnv2; one mEnv, one cEnv, and mEnv2; one mEnv+, one cEnv, and onemEnv2; one mEnv and two mEnv3; two mEnv and one mEnv3; one mEnv+ and twomEnv3; two mEnv+ and one mEnv3; one cEnv and two mEnv3; two cEnv and onemEnv3; one mEnv, one mEnv+, and one mEnv3; one mEnv, one cEnv, andmEnv3; one mEnv+, one cEnv, and one mEnv3; one mEnv, one mEnv2, and onemEnv3; one mEnv+, one mEnv2, and one mEnv3; or one cEnv, one mEnv2, andone mEnv3. Exemplary heterotrimers of the invention include Trimers 4-31in Table 1 below.

TABLE 1 Exemplary stabilized Env trimers Exemplary ConstituentPolypeptides Trimer Polypeptide 1 Polypeptide 2 Polypeptide 3 Trimer 1SEQ ID NO: 1 SEQ ID NO: 1 SEQ ID NO: 1 Trimer 2 SEQ ID NO: 2 SEQ ID NO:2 SEQ ID NO: 2 Trimer 3 SEQ ID NO: 3 SEQ ID NO: 3 SEQ ID NO: 3 Trimer 4SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 2 Trimer 5 SEQ ID NO: 1 SEQ ID NO:1 SEQ ID NO: 2 Trimer 6 SEQ ID NO: 1 SEQ ID NO: 3 SEQ ID NO: 3 Trimer 7SEQ ID NO: 1 SEQ ID NO: 1 SEQ ID NO: 3 Trimer 8 SEQ ID NO: 2 SEQ ID NO:3 SEQ ID NO: 3 Trimer 9 SEQ ID NO: 2 SEQ ID NO: 2 SEQ ID NO: 3 Trimer 10SEQ ID NO: 1 SEQ ID NO: 2 SEQ ID NO: 3 Trimer 11 SEQ ID NO: 1 SEQ ID NO:4 SEQ ID NO: 4 Trimer 12 SEQ ID NO: 1 SEQ ID NO: 1 SEQ ID NO: 4 Trimer13 SEQ ID NO: 2 SEQ ID NO: 4 SEQ ID NO: 4 Trimer 14 SEQ ID NO: 2 SEQ IDNO: 2 SEQ ID NO: 4 Trimer 15 SEQ ID NO: 3 SEQ ID NO: 4 SEQ ID NO: 4Trimer 16 SEQ ID NO: 3 SEQ ID NO: 3 SEQ ID NO: 4 Trimer 17 SEQ ID NO: 1SEQ ID NO: 2 SEQ ID NO: 4 Trimer 18 SEQ ID NO: 1 SEQ ID NO: 3 SEQ ID NO:4 Trimer 19 SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 4 Trimer 20 SEQ ID NO:1 SEQ ID NO: 5 SEQ ID NO: 5 Trimer 21 SEQ ID NO: 1 SEQ ID NO: 1 SEQ IDNO: 5 Trimer 22 SEQ ID NO: 2 SEQ ID NO: 5 SEQ ID NO: 5 Trimer 23 SEQ IDNO: 2 SEQ ID NO: 2 SEQ ID NO: 5 Trimer 24 SEQ ID NO: 3 SEQ ID NO: 5 SEQID NO: 5 Trimer 25 SEQ ID NO: 3 SEQ ID NO: 3 SEQ ID NO: 5 Trimer 26 SEQID NO: 1 SEQ ID NO: 2 SEQ ID NO: 5 Trimer 27 SEQ ID NO: 1 SEQ ID NO: 3SEQ ID NO: 5 Trimer 28 SEQ ID NO: 2 SEQ ID NO: 3 SEQ ID NO: 5 Trimer 29SEQ ID NO: 1 SEQ ID NO: 4 SEQ ID NO: 5 Trimer 30 SEQ ID NO: 2 SEQ ID NO:4 SEQ ID NO: 5 Trimer 31 SEQ ID NO: 3 SEQ ID NO: 4 SEQ ID NO: 5

Stabilized Gp140 Env Trimer Compositions of the Invention

Any one of the stabilized gp140 Env trimers of the invention, such asthose described above, can be included in compositions (e.g.,pharmaceutical compositions). Accordingly, the invention features acomposition including at least one of the stabilized gp140 Env trimersdescribed above (e.g., at least 2, 3, 4, 5, or more different types ofstabilized gp140 Env trimers may be included in a single composition orvaccine). For example, a composition including a homotrimer of mEnv ormEnv+ may additionally include an additional stabilized trimer form, forexample, an additional stabilized trimer form that includes three gp140polypeptides, wherein each of the gp140 polypeptides comprises an aminoacid sequence having at least 90% identity (e.g., at least 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to, or the sequence of,SEQ ID NO: 3 (cEnv).

The compositions may include a stabilized homotrimer including threemosaic Env1 polypeptides, for example, three polypeptides of mEnv orthree polypeptides of mEnv+ or three optimized clade C Env polypeptides,such as the cEnv polypeptide of SEQ ID NO: 3.

Alternatively, the compositions may also include a stabilizedheterotrimer. For example, the composition (e.g., a vaccine) may includeat least one stabilized heterotrimer that includes a combination of twodifferent mosaic Env1 sequences (e.g., one mEnv and two mEnv+; and twomEnv and one mEnv+). In some embodiments, the composition (e.g., avaccine) includes at least one stabilized heterotrimer that includescEnv and Env1 polypeptide (e.g., two mEnv and one cEnv; two mEnv+ andone cEnv; two cEnv and one mEnv; and two cEnv and one mEnv+). In otherembodiments, the compositions include at least one stabilizedheterotrimer that includes one cEnv and two different mosaic Env1polypeptides (e.g., one cEnv, one mEnv, and one mEnv+).

Optionally, the compositions may include at least one stabilizedheterotrimer having one or two constituent Env polypeptides including anamino acid sequence of SEQ ID NO: 4 (mosaic gp140 Env2, “mEnv2”) or SEQID NO: 5 (mosaic gp140 Env3, “mEnv3”). As noted above, preferably, mEnv2or mEnv3 may be, and is preferably, modified in a similar manner to thatof mEnv, mEnv+, or cEnv, which each possess a trimerization domain, asdiscussed above and depicted in FIGS. 1A-1C. Therefore, other vaccinesof the invention may include stabilized heterotrimers having thefollowing constituent polypeptides: one mEnv and two mEnv2; two mEnv andone mEnv2; one mEnv+ and two mEnv2; two mEnv+ and one mEnv2; one cEnvand two mEnv2; two cEnv and one mEnv2; one mEnv, one mEnv+, and onemEnv2; one mEnv, one cEnv, and mEnv2; one mEnv+, one cEnv, and onemEnv2; one mEnv and two mEnv3; two mEnv and one mEnv3; one mEnv+ and twomEnv3; two mEnv+ and one mEnv3; one cEnv and two mEnv3; two cEnv and onemEnv3; one mEnv, one mEnv+, and one mEnv3; one mEnv, one cEnv, andmEnv3; one mEnv+, one cEnv, and one mEnv3; one mEnv, one mEnv2, and onemEnv3; one mEnv+, one mEnv2, and one mEnv3; or one cEnv, one mEnv2, andone mEnv3.

Any one of the compositions of the invention may further include apharmaceutically acceptable carrier, excipient, or diluent, and/or anadjuvant.

Stabilized Gp140 Env Trimer Vaccines of the Invention

The invention features vaccines including at least one of thecompositions of the invention described herein and above. The vaccinemay be used for treating or reducing the risk of a humanimmunodeficiency virus (HIV) infection in a subject in need thereof. Forexample, the vaccine may elicit production of neutralizing anti-HIVantisera (e.g., neutralizing anti-HIV-1 antisera) after administrationto the subject. The anti-HIV antisera may also be able to neutralize HIV(e.g., HIV-1), for example, selected from any one or more of clade A,clade B, and clade C.

Nucleic Acid Molecules of the Invention

In some embodiments, the vaccines of the invention include one or morenucleic acid molecules of the invention, such as a nucleic acid moleculehaving a nucleotide sequence that encodes a gp140 polypeptide, in whichthe gp140 polypeptide includes (a) an amino acid sequence having atleast 95% identity (e.g., 96%, 97%, 98%, 99%, or 100% identity) to SEQID NO: 1, (b) an amino acid sequence having at least 95% identity (e.g.,96%, 97%, 98%, 99%, or 100% identity) to SEQ ID NO: 2, and/or (c) anamino acid sequence having the sequence of SEQ ID NO: 3, (d) an aminoacid sequence having the sequence of SEQ ID NO:4, (e) an amino acidsequence having the sequence of SEQ ID NO:5 and/or combinations thereof.As discussed below, vectors (e.g., viral vectors, such as an adenovirusor poxvirus vector) of the invention can include one or more of thesenucleic acid molecules. Accordingly, vaccines of the invention mayinclude one or more of these vectors. The stabilized gp140 Env trimerpolypeptides of the invention, as well as vaccines, nucleic acids, andvectors that incorporate one or more optimized gp140 Env polypeptides,can be recombinantly expressed in a cell or organism, or can be directlyadministered to a subject (e.g., a human) infected with, or at risk ofbecoming infected with, HIV (e.g., HIV-1).

Vectors of the Invention

As noted above, the invention features vectors including one or more ofthe nucleic acid molecules of the invention. The vector can be, forexample, a carrier (e.g., a liposome), a plasmid, a cosmid, a yeastartificial chromosome, or a virus (e.g., an adenovirus vector or apoxvirus vector) that includes one or more of the nucleic acid moleculesof the invention.

An adenovirus vector of the invention can be derived from a recombinantadenovirus serotype 11 (Ad11), adenovirus serotype 15 (Ad15), adenovirusserotype 24 (Ad24), adenovirus serotype 26 (Ad26), adenovirus serotype34 (Ad34), adenovirus serotype 35 (Ad35), adenovirus serotype 48 (Ad48),adenovirus serotype 49 (Ad49), adenovirus serotype 50 (Ad50), Pan9(AdC68), or a chimeric variant thereof (e.g., adenovirus serotype 5HVR48 (Ad5HVR48)). A poxvirus vector of the invention may be derived,for example, from modified vaccinia virus Ankara (MVA). These vectorscan include additional nucleic acid sequences from several sources.

Vectors of the invention can be constructed using any recombinantmolecular biology technique known in the art. The vector, upontransfection or transduction of a target cell or organism, can beextrachromosomal or integrated into the host cell chromosome. Thenucleic acid component of a vector can be in single or multiple copynumber per target cell, and can be linear, circular, or concatamerized.The vectors can also include internal ribosome entry site (IRES)sequences to allow for the expression of multiple peptide or polypeptidechains from a single nucleic acid transcript (e.g., a polycistronicvector, e.g., a bi- or tri-cistronic vector).

Vectors of the invention can also include gene expression elements thatfacilitate the expression of the encoded polypeptide(s) of the invention(e.g., SEQ ID NOs: 1 (mEnv), 2 (mEnv+), and/or 3 (cEnv) or polypeptideshaving amino acids sequences with at least 90%, 91%, 92$, 93&, 94%, 95%,96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1 or 2). Geneexpression elements include, but are not limited to, (a) regulatorysequences, such as viral transcription promoters and their enhancerelements, such as the SV40 early promoter, Rous sarcoma virus LTR, andMoloney murine leukemia virus LTR; (b) splice regions andpolyadenylation sites such as those derived from the SV40 late region;and (c) polyadenylation sites such as in SV40. Also included are plasmidorigins of replication, antibiotic resistance or selection genes,multiple cloning sites (e.g., restriction enzyme cleavage loci), andother viral gene sequences (e.g., sequences encoding viral structural,functional, or regulatory elements, such as the HIV long terminal repeat(LTR)).

Exemplary vectors are described below.

Adenovirus Vectors

Recombinant adenoviruses offer several significant advantages for use asvectors for the expression of, for example, one or more of the optimizedgp140 Env polypeptides of the invention. The viruses can be prepared tohigh titer, can infect non-replicating cells, and can conferhigh-efficiency transduction of target cells ex vivo following contactwith a target cell population. Furthermore, adenoviruses do notintegrate their DNA into the host genome. Thus, their use as expressionvectors has a reduced risk of inducing spontaneous proliferativedisorders. In animal models, adenoviral vectors have generally beenfound to mediate high-level expression for approximately one week. Theduration of transgene expression (expression of a nucleic acid moleculeof the invention) can be prolonged by using cell or tissue-specificpromoters. Other improvements in the molecular engineering of theadenovirus vector itself have produced more sustained transgeneexpression and less inflammation. This is seen with so-called “secondgeneration” vectors harboring specific mutations in additional earlyadenoviral genes and “gutless” vectors in which virtually all the viralgenes are deleted utilizing a Cre-Lox strategy (Engelhardt et al., Proc.Natl. Acad. Sci. USA 91:6196 (1994) and Kochanek et al., Proc. Natl.Acad. Sci. USA 93:5731 (1996), each herein incorporated by reference).

The rare serotype and chimeric adenoviral vectors disclosed inInternational Patent Application Publications WO 2006/040330 and WO2007/104792, each incorporated by reference herein, are particularlyuseful as vectors of the invention. For example, recombinant adenovirusserotype 11 (Ad11), adenovirus serotype 15 (Ad15), adenovirus serotype24 (Ad24), adenovirus serotype 26 (Ad26), adenovirus serotype 34 (Ad34),adenovirus serotype 35 (Ad35), adenovirus serotype 48 (Ad48), adenovirusserotype 49 (Ad49), adenovirus serotype 50 (Ad50), Pan9 (AdC68), or achimeric variant thereof (e.g., adenovirus serotype 5 HVR48 (Ad5HVR48)can encode and/or deliver one or more of the optimized gp140 Envpolypeptides of the invention to facilitate formation and presentationof gp140 Env trimer formation. In some embodiments, one or morerecombinant adenovirus vectors can be administered to the subject inorder to express gp140 Env polypeptides for formation of stabilizedtrimers of the invention.

Adeno-Associated Virus (AAV) Vectors

Adeno-associated viruses (AAV), derived from non-pathogenicparvoviruses, can also be used to facilitate delivery and/or expressionof one or more of the optimized gp140 Env polypeptides of the inventionas these vectors evoke almost no anti-vector cellular immune response,and produce transgene expression lasting months in most experimentalsystems.

Stabilized trimers of the invention may be produced upon expression ofthe gp140 Env polypeptides described herein using an AAV vector.

Retrovirus Vectors

Retroviruses are useful for the expression of optimized gp140 Envpolypeptides of the invention. Unlike adenoviruses, the retroviralgenome is based in RNA. When a retrovirus infects a cell, it willintroduce its RNA together with several enzymes into the cell. The viralRNA molecules from the retrovirus will produce a double-stranded DNAcopy, called a provirus, through a process called reverse transcription.Following transport into the cell nucleus, the proviral DNA isintegrated in a host cell chromosome, permanently altering the genome ofthe transduced cell and any progeny cells that may derive from thiscell. The ability to permanently introduce a gene into a cell ororganism is the defining characteristic of retroviruses used for genetherapy. Retroviruses include lentiviruses, a family of virusesincluding human immunodeficiency virus (HIV) that includes severalaccessory proteins to facilitate viral infection and proviralintegration. Current, “third-generation,” lentiviral vectors featuretotal replication incompetence, broad tropism, and increased genetransfer capacity for mammalian cells (see, e.g., Mangeat and Trono,Human Gene Therapy 16(8):913 (2005) and Wiznerowicz and Trono, TrendsBiotechnol. 23(1):42 (2005), each herein incorporated by reference).

Stabilized trimers of the invention may be produced upon expression ofthe gp140 Env polypeptides described herein using a retrovirus vector.

Other Viral Vectors

Besides adenoviral and retroviral vectors, other viral vectors andtechniques are known in the art that can be used to facilitate deliveryand/or expression of one or more of the optimized gp140 Env polypeptidesof the invention in a cell (e.g., a blood cell, such as a lymphocyte) orsubject (e.g., a human) in order to promote formation of the trimers ofthe invention. These viruses include poxviruses (e.g., vaccinia virusand modified vaccinia virus Ankara (MVA); see, e.g., U.S. Pat. Nos.4,603,112 and 5,762,938, each incorporated by reference herein),herpesviruses, togaviruses (e.g., Venezuelan Equine Encephalitis virus;see, e.g., U.S. Pat. No. 5,643,576, incorporated by reference herein),picornaviruses (e.g., poliovirus; see, e.g., U.S. Pat. No. 5,639,649,incorporated by reference herein), baculoviruses, and others describedby Wattanapitayakul and Bauer (Biomed. Pharmacother. 54:487 (2000),incorporated by reference herein).

Naked DNA and Oligonucleotides

Naked DNA or oligonucleotides encoding one or more of the optimizedgp140 Env polypeptides of the invention can also be used to expressthese polypeptides in a cell or a subject (e.g., a human) in order topromote formation of the trimers of the invention. See, e.g., Cohen,Science 259:1691-1692 (1993); Fynan et al., Proc. Natl. Acad. Sci. USA,90:11478 (1993); and Wolff et al., BioTechniques 11:474485 (1991), eachherein incorporated by reference. This is the simplest method ofnon-viral transfection. Efficient methods for delivery of naked DNAexist, such as electroporation and the use of a “gene gun,” which shootsDNA-coated gold particles into a cell using high pressure gas andcarrier particles (e.g., gold).

Lipoplexes and Polyplexes

To improve the delivery of a nucleic acid encoding one or more of theoptimized gp140 Env polypeptides of the invention into a cell or subjectin order to promote formation of the trimers of the invention,lipoplexes (e.g., liposomes) and polyplexes can be used to protect thenucleic acid from undesirable degradation during the transfectionprocess. The nucleic acid molecules can be covered with lipids in anorganized structure like a micelle or a liposome. When the organizedstructure is complexed with the nucleic acid molecule it is called alipoplex. There are three types of lipids: anionic (negatively-charged),neutral, or cationic (positively-charged). Lipoplexes that utilizecationic lipids have proven utility for gene transfer. Cationic lipids,due to their positive charge, naturally complex with thenegatively-charged nucleic acid. Also as a result of their charge theyinteract with the cell membrane, endocytosis of the lipoplex occurs, andthe nucleic acid is released into the cytoplasm. The cationic lipidsalso protect against degradation of the nucleic acid by the cell.

Complexes of polymers with nucleic acids are called polyplexes. Mostpolyplexes consist of cationic polymers and their production isregulated by ionic interactions. One large difference between themethods of action of polyplexes and lipoplexes is that polyplexes cannotrelease their nucleic acid load into the cytoplasm, so, to this end,co-transfection with endosome-lytic agents (to lyse the endosome that ismade during endocytosis) such as inactivated adenovirus must occur.However, this is not always the case; polymers such as polyethyleniminehave their own method of endosome disruption as does chitosan andtrimethylchitosan.

Exemplary cationic lipids and polymers that can be used in combinationwith one or more of the nucleic acid molecules encoding one or more ofthe optimized gp140 Env polypeptides of the invention to form lipoplexesor polyplexes include, but are not limited to, polyethylenimine,lipofectin, lipofectamine, polylysine, chitosan, trimethylchitosan, andalginate.

Hybrid Methods

Several hybrid methods of gene transfer combine two or more techniques.Virosomes, for example, combine lipoplexes (e.g., liposomes) with aninactivated virus. This approach has been shown to result in moreefficient gene transfer in respiratory epithelial cells compared toeither viral or liposomal methods alone. Other methods involve mixingother viral vectors with cationic lipids or hybridizing viruses. Each ofthese methods can be used to facilitate transfer of one or more of thenucleic acid molecules of the invention encoding one or more of theoptimized gp140 Env polypeptides of the invention into a cell or subjectin order to promote formation of the trimers of the invention.

Dendrimers

Dendrimers may be also be used to transfer one or more of the nucleicacid molecules of the invention encoding one or more of the optimizedgp140 Env polypeptides of the invention into a cell or subject in orderto promote formation of the trimers of the invention. A dendrimer is ahighly branched macromolecule with a spherical shape. The surface of theparticle may be functionalized in many ways, and many of the propertiesof the resulting construct are determined by its surface. In particularit is possible to construct a cationic dendrimer (i.e., one with apositive surface charge). When in the presence of genetic material(e.g., a nucleic acid molecule), charge complimentarily leads to atemporary association of the nucleic acid with the cationic dendrimer.On reaching its destination the dendrimer-nucleic acid complex is thentaken into the cell via endocytosis.

Methods of Treatment Using the Compositions of the Invention

In Vivo Administration

The invention features methods for the in vivo administration of atherapeutically effective amount of one or more of the compositions(i.e., vaccines, vectors, stabilized trimer(s), nucleic acids,polypeptides, stabilized trimer, or other composition thereof describedherein) of the invention to a subject (e.g., a human, e.g., a humaninfected with HIV or a human at risk of an HIV infection) in needthereof. Upon administering one or more of the compositions of theinvention to the subject, the stabilized trimers of the invention canelicit protective or therapeutic immune responses (e.g., cellular orhumoral immune responses, e.g., neutralizing anti-HIV antiseraproduction, e.g., anti-HIV antisera that neutralizes HIV selected fromclade A, clade B, and/or clade C HIV) directed against the viralimmunogens.

The method may be used to treat or reduce the risk of an HIV infectionin a subject in need thereof. The subject may be infected with HIV ormay be at risk of exposure to HIV. The compositions of the invention canbe administered to a subject infected with HIV to treat AIDS. Examplesof symptoms of diseases caused by a viral infection, such as AIDS, thatcan be treated using the compositions of the invention include, forexample, fever, muscle aches, coughing, sneezing, runny nose, sorethroat, headache, chills, diarrhea, vomiting, rash, weakness, dizziness,bleeding under the skin, in internal organs, or from body orifices likethe mouth, eyes, or ears, shock, nervous system malfunction, delirium,seizures, renal (kidney) failure, personality changes, neck stiffness,dehydration, seizures, lethargy, paralysis of the limbs, confusion, backpain, loss of sensation, impaired bladder and bowel function, andsleepiness that can progress into coma or death. These symptoms, andtheir resolution during treatment, may be measured by, for example, aphysician during a physical examination or by other tests and methodsknown in the art.

In cases in which the subject is infected with HIV, the method may beused to reduce an HIV-mediated activity (e.g., infection, fusion (e.g.,target cell entry and/or syncytia formation), viral spread, etc.) and/orto decrease HIV titer in the subject. HIV-mediated activity and/or HIVtiter may be decreased, for example, by 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% ormore compared to that of a control subject (e.g., an untreated subjector a subject treated with a placebo).

One or more of the compositions of the invention may also beadministered in the form of a vaccine for prophylactic treatment of asubject (e.g., a human) at risk of an HIV infection.

The compositions utilized in the methods described herein can beformulated, for example, for administration intramuscularly,intravenously, intradermally, percutaneously, intraarterially,intraperitoneally, intralesionally, intracranially, intraarticularly,intraprostatically, intrapleurally, intratracheally, intranasally,intravitreally, intravaginally, intrarectally, topically,intratumorally, peritoneally, subcutaneously, subconjunctivally,intravesicularlly, mucosally, intrapericardially, intraumbilically,intraocularly, orally, topically, locally, by inhalation, by injection,by infusion, by continuous infusion, by localized perfusion bathingtarget cells directly, by catheter, by lavage, by gavage, in cremes, orin lipid compositions.

The preferred method of administration can vary depending on variousfactors (e.g., the components of the composition being administered andthe severity of the condition being treated). Formulations suitable fororal or nasal administration may consist of liquid solutions, such as aneffective amount of the composition dissolved in a diluent (e.g., water,saline, or PEG-400), capsules, sachets, tablets, or gels, eachcontaining a predetermined amount of the chimeric Ad5 vector compositionof the invention. The pharmaceutical composition may also be an aerosolformulation for inhalation, for example, to the bronchial passageways.Aerosol formulations may be mixed with pressurized, pharmaceuticallyacceptable propellants (e.g., dichlorodifluoromethane, propane, ornitrogen). In particular, administration by inhalation can beaccomplished by using, for example, an aerosol containing sorbitantrioleate or oleic acid, for example, together withtrichlorofluoromethane, dichlorofluoromethane,dichlorotetrafluoroethane, or any other biologically compatiblepropellant gas.

Immunogenicity of the composition of the invention may be significantlyimproved if it is co-administered with an immunostimulatory agent oradjuvant. Suitable adjuvants well-known to those skilled in the artinclude, for example, aluminum phosphate, aluminum hydroxide, QS21, QuilA (and derivatives and components thereof), calcium phosphate, calciumhydroxide, zinc hydroxide, glycolipid analogs, octodecyl esters of anamino acid, muramyl dipeptides, polyphosphazene, lipoproteins, ISCOMmatrix, DC-Chol, DDA, cytokines, and other adjuvants and derivativesthereof.

Compositions according to the invention described herein may beformulated to release the composition immediately upon administration(e.g., targeted delivery) or at any predetermined time period afteradministration using controlled or extended release formulations.Administration of the composition in controlled or extended releaseformulations is useful where the composition, either alone or incombination, has (i) a narrow therapeutic index (e.g., the differencebetween the plasma concentration leading to harmful side effects ortoxic reactions and the plasma concentration leading to a therapeuticeffect is small; generally, the therapeutic index, TI, is defined as theratio of median lethal dose (LD₅₀) to median effective dose (ED₅₀));(ii) a narrow absorption window at the site of release (e.g., thegastro-intestinal tract); or (iii) a short biological half-life, so thatfrequent dosing during a day is required in order to sustain atherapeutic level.

Many strategies can be pursued to obtain controlled or extended releasein which the rate of release outweighs the rate of metabolism of thepharmaceutical composition. For example, controlled release can beobtained by the appropriate selection of formulation parameters andingredients, including, for example, appropriate controlled releasecompositions and coatings. Suitable formulations are known to those ofskill in the art. Examples include single or multiple unit tablet orcapsule compositions, oil solutions, suspensions, emulsions,microcapsules, microspheres, nanoparticles, patches, and liposomes.

The compositions of the invention may be administered to providepre-infection prophylaxis or after a subject has been diagnosed with anHIV infection or a disease with an etiology traceable to an HIVinfection (e.g., AIDS). The composition may be administered, forexample, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 35, 40, 45, 50, 55,or 60 minutes, 2, 4, 6, 10, 15, or 24 hours, 2, 3, 5, or 7 days, 2, 4, 6or 8 weeks, or even 3, 4, or 6 months pre-infection or pre-diagnosis, ormay be administered to the subject 15-30 minutes or 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 12, 20, 24, 48, or 72 hours, 2, 3, 5, or 7 days, 2, 4, 6 or 8weeks, 3, 4, 6, or 9 months, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20years or longer post-diagnosis or post-infection to HIV. The subject canbe administered a single dose of the composition(s) (or, e.g., 2, 3, 4,5, 6, 7, 8, 9, 10, or more doses) or the subject can be administered atleast one dose (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more doses)daily, weekly, monthly, or yearly. The administration period may bedefined (e.g., 1-4 weeks, 1-12 months, 1-20 years) or may be for thelife of the subject. The composition(s) may also be administered to saidsubject as a prime or a boost composition or in a prime-boost regimen.In a preferred embodiment, the composition (e.g., vaccine) of theinvention is administered as a boost following administration of anadditional composition (e.g., vaccine) as a prime, where the primeincludes at least a first vector including a first nucleic acid moleculethat encodes a polypeptide having at least 85% amino acid sequenceidentity (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identity) to, or the sequence of, SEQ ID NO: 6, andoptionally a second vector including a second nucleic acid molecule thatencodes a polypeptide having at least 85% identity (e.g., 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity)to, or the sequence of, SEQ ID NO: 7. The boost in this regimen includesone or more of the composition(s) of the invention (e.g., any one of thestabilized trimers, the compositions, the vaccines, the nucleic acidmolecules, and/or the vectors of the invention). In still otherembodiments, the prime includes at least a first vector including anucleic acid molecule that encodes a polypeptide having the sequence ofany one of SEQ ID NOs: 8-32. Alternatively, the composition (e.g.,vaccine) of the invention is administered as a prime. In someembodiments where the composition of the invention is administered as aprime, a different vaccine (e.g., a vaccine including at least a firstvector including a first nucleic acid molecule that encodes apolypeptide having at least 85% amino acid sequence identity (e.g., 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity) to, or the sequence of, SEQ ID NO: 6, and optionally a secondvector including a second nucleic acid molecule that encodes apolypeptide having at least 85% identity (e.g., 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity) to, or thesequence of, SEQ ID NO: 7; or a vaccine including at least a firstvector including a nucleic acid molecule that encodes a polypeptidehaving the sequence of any one of SEQ ID NOs: 8-32) is administered as aboost.

When treating disease (e.g., AIDS), the compositions of the inventionmay be administered to the subject either before the occurrence ofsymptoms or a definitive diagnosis or after diagnosis or symptoms becomeevident. For example, the composition may be administered, for example,immediately after diagnosis or the clinical recognition of symptoms or2, 4, 6, 10, 15, or 24 hours, 2, 3, 5, or 7 days, 2, 4, 6 or 8 weeks, oreven 3, 4, or 6 months after diagnosis or detection of symptoms.

The compositions may be sterilized by conventional sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation may be administered in powder form or combined with asterile aqueous carrier prior to administration. The pH of thepreparations typically will be between 3 and 11, more preferably between5 and 9 or between 6 and 8, and most preferably between 7 and 8, such as7 to 7.5. The resulting compositions in solid form may be packaged inmultiple single dose units, each containing a fixed amount of any one ormore of the optimized gp140 Env nucleic acids required to supportformation of one or more of the stabilized trimers of the inventionand/or one or more of the stabilized trimers of the invention of theinvention and, if desired, one or more immunomodulatory agents, such asin a sealed package of tablets or capsules, or in a suitable dry powderinhaler (DPI) capable of administering one or more doses.

Dosages

The dose of a composition of the invention (e.g., a vaccine includingone or more of the stabilized gp140 Env trimers of the invention) or thenumber of treatments using a composition of the invention may beincreased or decreased based on the severity of, occurrence of, orprogression of, the HIV infection and/or disease related to the HIVinfection (e.g., AIDS) in the subject (e.g., based on the severity ofone or more symptoms of HIV infection/AIDS described above).

The stabilized gp140 Env trimer compositions of the invention can beadministered in a therapeutically effective amount that provides animmunogenic and/or protective effect against HIV or target protein ofHIV (e.g., gp140). The subject may, for example, be administeredpolypeptide compositions of the invention (e.g., stabilized gp140 Envtrimers of the invention) in a non-vectored composition. The polypeptidecomposition administered may include between approximately 1 μg and 1 mgof stabilized Env trimers, and more preferably between 50 μg and 300 μgof stabilized Env trimers of the invention.

Alternatively, the subject may be administered, in the form of a viralvector, at least about 1×10³ viral particles (vp)/dose or between 1×10¹and 1×10¹⁴ vp/dose, preferably between 1×10³ and 1×10¹² vp/dose, andmore preferably between 1×10⁵ and 1×10¹¹ vp/dose.

Viral particles include nucleic acid molecules encoding one or more ofthe optimized gp140 Env polypeptides of the invention and are surroundedby a protective coat (a protein-based capsid with hexon and fiberproteins). Viral particle number can be measured based on, for example,lysis of vector particles, followed by measurement of the absorbance at260 nm (see, e.g,. Steel, Curr. Opin. Biotech., 1999).

The dosage administered depends on the subject to be treated (e.g., theage, body weight, capacity of the immune system, and general health ofthe subject being treated), the form of administration (e.g., as a solidor liquid), the manner of administration (e.g., by injection,inhalation, dry powder propellant), and the cells targeted (e.g.,epithelial cells, such as blood vessel epithelial cells, nasalepithelial cells, or pulmonary epithelial cells). The composition ispreferably administered in an amount that provides a sufficient level ofthe stabilized gp140 Env trimer gene product (e.g., a level ofstabilized gp140 Env trimer that elicits an immune response withoutundue adverse physiological effects in the subject caused by theimmunogenic trimer).

In addition, single or multiple administrations of the compositions ofthe present invention may be given (pre- or post-infection and/or pre-or post-diagnosis) to a subject (e.g., one administration oradministration two or more times). For example, subjects who areparticularly susceptible to, for example, HIV infection may requiremultiple treatments to establish and/or maintain protection against thevirus. Levels of induced immunity provided by the pharmaceuticalcompositions described herein can be monitored by, for example,measuring amounts of neutralizing anti-HIV secretory and serumantibodies. The dosages may then be adjusted or repeated as necessary totrigger the desired level of immune response. For example, the immuneresponse triggered by a single administration (prime) of a compositionof the invention may not be sufficiently potent and/or persistent toprovide effective protection. Accordingly, in some embodiments, repeatedadministration (boost), such that a prime-boost regimen is established,may significantly enhance humoral and cellular responses to the antigenof the composition.

Alternatively, as applies to recombinant therapy, the efficacy oftreatment can be determined by monitoring the level of the one or moreoptimized gp140 Env trimers expressed by or present in a subject (e.g.,a human) following administration of the compositions of the invention.For example, the blood or lymph of a subject can be tested for theimmunogenic trimer(s) using, for example, standard assays known in theart (see, e.g., Human Interferon-Alpha Multi-Species ELISA kit (ProductNo. 41105) and the Human Interferon-Alpha Serum Sample kit (Product No.41110) from Pestka Biomedical Laboratories (PBL), Piscataway, N.J.).

A single dose of one or more of the compositions of the invention mayachieve protection, pre-infection or pre-diagnosis. In addition, asingle dose administered post-infection or post-diagnosis can functionas a treatment according to the present invention.

A single dose of one or more of the compositions of the invention canalso be used to achieve therapy in subjects being treated for a disease.Multiple doses (e.g., 2, 3, 4, 5, or more doses) can also beadministered, in necessary, to these subjects.

Carriers, Excipients, Diluents

Therapeutic formulations of the compositions of the invention (e.g.,vaccines, vectors, stabilized trimer(s), nucleic acid molecules, etc.)may be prepared using standard methods known in the art by mixing theactive ingredient having the desired degree of purity with optionalphysiologically acceptable carriers, excipients, or stabilizers(Remington's Pharmaceutical Sciences (20^(th) edition), ed. A. Gennaro,2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.). Acceptablecarriers, include saline, or buffers such as phosphate, citrate andother organic acids; antioxidants including ascorbic acid; low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone, amino acids such as glycine, glutamine,asparagines, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, PLURONICS™, or PEG.

Optionally, but preferably, the formulation contains a pharmaceuticallyacceptable salt, preferably sodium chloride, and preferably at aboutphysiological concentrations. Optionally, the formulations of theinvention can contain a pharmaceutically acceptable preservative. Insome embodiments the preservative concentration ranges from 0.1 to 2.0%,typically v/v. Suitable preservatives include those known in thepharmaceutical arts. Benzyl alcohol, phenol, m-cresol, methylparaben,and propylparaben are preferred preservatives. Optionally, theformulations of the invention can include a pharmaceutically acceptablesurfactant at a concentration of 0.005 to 0.02%.

Adjuvants

Any one of the compositions of the invention (e.g., vaccines, vectors,stabilized trimer(s), nucleic acid molecules, etc.) can be formulated toinclude, be administered concurrently with, and/or be administered inseries with one or more pharmaceutically acceptable adjuvants toincrease the immunogenicity of the composition (e.g., uponadministration to a subject in need thereof, e.g., a subject infectedwith HIV or at risk of an HIV infection). Adjuvants approved for humanuse include aluminum salts (alum). These adjuvants have been useful forsome vaccines including hepatitis B, diphtheria, polio, rabies, andinfluenza. Other useful adjuvants include Complete Freund's Adjuvant(CFA), Incomplete Freund's Adjuvant (IFA), muramyl dipeptide (MDP),synthetic analogues of MDP,N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-alanine-2-[1,2-dipalmitoyl-s-gly-cero-3-(hydroxyphosphoryloxy)]ethylamide(MTP-PE) and compositions containing a metabolizable oil and anemulsifying agent, wherein the oil and emulsifying agent are present inthe form of an oil-in-water emulsion having oil droplets substantiallyall of which are less than one micron in diameter.

Ex Vivo Transfection and Transduction

The present invention also provides for the ex vivo transfection ortransduction of cells, followed by administration of these cells backinto a subject (e.g., human) to allow for the expression of one or moreof the optimized gp140 Env polypeptides of the invention that haveimmunogenic properties. In one embodiment, the cells are autologous tothe treated subject. Cells can be transfected or transduced ex vivowith, for example, one or more vectors of the invention to allow for thetemporal or permanent expression of one or more of the optimized gp140Env polypeptides in the treated subject. Upon administering thesemodified cells to the subject, the one or more vectors of the inventionwill be expressed, eliciting protective or therapeutic immune responses(e.g., cellular or humoral immune responses, e.g., production ofneutralizing anti-HIV antisera) directed against the gp140 immunogenictrimer or trimers that form.

Cells that can be isolated and transfected or transduced ex vivoaccording to the methods of invention include, but are not limited to,blood cells, skin cells, fibroblasts, endothelial cells, skeletal musclecells, hepatocytes, prostate epithelial cells, and vascular endothelialcells. Stem cells are also appropriate cells for transduction ortransfection with a vector of the invention. Totipotent, pluripotent,multipotent, or unipotent stem cells, including bone marrow progenitorcells and hematopoietic stem cells (HSC), can be isolated andtransfected or transduced with, for example, a vector of the invention,and administered to a subject according to the methods of the invention.

The method of transfection or transduction has a strong influence on thestrength and longevity of protein expression (e.g., stabilized gp140trimer expression) in the transfected or transduced cell, andsubsequently, in the subject (e.g., human) receiving the cell. Thepresent invention provides vectors that are temporal (e.g., adenoviralvectors) or long-lived (e.g., retroviral vectors) in nature. Regulatorysequences (e.g., promoters and enhancers) are known in the art that canbe used to regulate protein expression. The type of cell beingtransfected or transduced also has a strong bearing on the strength andlongevity of protein expression. For example, cell types with high ratesof turnover can be expected to have shorter periods of proteinexpression.

Kits

The invention provides kits that include a pharmaceutical compositioncontaining a vaccine, vector, stabilized trimer, or optimized viralpolypeptide of the invention, and a pharmaceutically-acceptable carrier,in a therapeutically effective amount for preventing or treating a viralinfection. The kits include instructions to allow a clinician (e.g., aphysician or nurse) to administer the composition contained therein.

Preferably, the kits include multiple packages of the single-dosepharmaceutical composition(s) containing an effective amount of avaccine, vector, stabilized trimer, or optimized viral polypeptide ofthe invention. Optionally, instruments or devices necessary foradministering the pharmaceutical composition(s) may be included in thekits. For instance, a kit of this invention may provide one or morepre-filled syringes containing an effective amount of a vaccine, vector,stabilized trimer, or optimized viral polypeptide of the invention.Furthermore, the kits may also include additional components such asinstructions or administration schedules for a patient infected with orat risk of being infected with a virus to use the pharmaceuticalcomposition(s) containing a vaccine, vector, stabilized trimer, oroptimized viral polypeptide of the invention.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the compositions, methods,and kits of the present invention without departing from the spirit orscope of the invention. Thus, it is intended that the present inventioncover the modifications and variations of this invention provided theycome within the scope of the appended claims and their equivalents.

EXAMPLES

The present invention is illustrated by the following examples, whichare in no way intended to be limiting of the invention.

Example 1. Materials and Methods Western Blot Immunodetection

Volumes containing 10-μg equivalents of DNA expression vectors pVRC8400empty, pVRC8400 mosaic gp140 version-1 (expression vector for apolypeptide including the amino acid sequence of SEQ ID NO: 1), orpVRC8400 mosaic gp140 version-2 (expression vector for a polypeptideincluding the amino acid sequence of SEQ ID NO: 2) were each made up to100 μl with Dulbeco's Modified Eagle Medium (DMEM; Invitrogen). 40 μl ofLipofectamine (Invitrogen) transfection reagent was then added 60 μlDMEM and 100 μl of this mix added to each DNA vector followed by gentleagitation and incubation at room temperature for 30 minutes. 293T cellsgrown to approximately 70-80% confluency in T-25 flasks were washed oncewith 2.5 ml DMEM, 2.3 ml of DMEM added followed by 200 μlDNA/Lipofectamine mix. Cells were then incubated at 37° C., 10% CO₂ for48 hours. 48 hours post-transfection, 0.5 ml of supernatant from eachT-25 flask was harvested, briefly spun and 20 μl placed in a fresheppendorf tube. 5 μl of 5× reducing sample buffer (Pierce) was added toeach tube, each sample heated for 5 minutes at 100° C. and then place onice to cool. 20 μl of each sample was loaded on a 4-15% pre-castSDS-PAGE (Biorad), and the gel run at 150V for approximately 70 minutes.Transfer of protein from gel to membrane was performed using the iblotdry blotting system (Invitrogen) as per vendor protocol using PVDF geltransfer stacks. Membrane blocking was performed overnight at 4° C. in20 ml of PBS-T Block (i.e., Dulbeco's phosphate buffered saline(Invitrogen), containing 0.2% V/V Tween 20 (Sigma) and 5% W/V non-fatmilk powder) on an orbital shaker. 10 μl of monoclonal HRP conjugatedanti-His tag antibody (Qiagen) was then added to 20 ml PBS-T Block(1:2000 dilution) followed by incubation on an orbital shaker at roomtemperature for 1 hour. Membranes were washed 5 times in PBS-T block,membranes touch dried on absorbent paper to remove excess block, and fordetection, Amersham ECL Plus Western Blotting Detection System (GEHealthcare) was utilized.

Roller Bottle Transfection and Protein Purification

DMEM growth media supplemented with 10% Fetal Bovine Serum (FBS) wasused to grow 293T to confluence in Cell Bind® roller bottles (Corning),growth media removed, followed by addition of 250 ml of pre-warmedFreestyle 293 expression medium (Invitrogen) and incubation for 2 hoursat 37° C., 5% CO₂. 250 μg of DNA expression vector pVRC8400 mosaic gp140version-2 was mixed with 320 μl of polyethylenimine (PEI) (1 mg/ml)added to 20 ml of room temperature freestyle 293 medium, incubated atroom temperature for 20 minutes and then added in each roller bottlefollowed by incubation for 6 days in 37° C., 5% CO₂. The cellsupernatant was harvested at 6 days after medium change. TheHistidine-tagged optimized mosaic gp140 Env version-2 protein, includingSEQ ID NO: 2, was purified by Ni-NTA (Qiagen) followed by size-exclusionchromatography. Briefly, after a clarifying spin and the addition ofimidazole to the final concentration of 10 mM, the cell supernatant wasloaded onto a nickel column at a flow rate of 0.8 mL/min and was washedwith 20 mM imidazole in PBS followed by further washing with 40 mMimidazole in PBS. The protein then was eluted with 300 mM imidazole inPBS. The fractions containing the purified protein were pooled,concentrated, and further purified by gel-filtration chromatography onSuperose 6 (GE Healthcare) in a column running buffer containing 25 mMTris (pH 7.5) and 150 mM NaCl. The purified proteins were concentrated,frozen in liquid nitrogen, and stored at −80° C.

Animals and Immunizations

Outbred female Hartley guinea pigs (Elm Hill Labs) were housed at theAnimal Research Facility of Beth Israel Deaconess Medical Center underprotocols approved by the Institutional Animal Care and Use Committee.Guinea pigs were immunized by bilateral intramuscular injections in theupper quadriceps with clade C gp140 Env polypeptide (i.e., homotrimer ofthree molecules including the amino acid sequence of SEQ ID NO: 3),mosaic gp140 Env (i.e., homotrimer of three molecules including theamino acid sequence of SEQ ID NO: 1), or a clade C gp140 Env/mosaicgp140 Env mixture (100 μg/animal) at 4-week intervals (weeks 0, 4, and8) using 500 μl of a dual adjuvant combination comprising 15% (v/v)oil-in-water Emulsigen (MVP Laboratories)/PBS and 50 μg ofimmunostimulatory di-nucleotide CpG DNA (5′-TCGTCGTTGTCGTTTTGTCGTT-3′)(Midland Reagent Company). The clade C gp140 Env/mosaic gp140 Envmixture contained 50 μg of each protein. Serum samples were obtainedfrom the vena cava of anesthetized animals 4 weeks after eachimmunization.

Neutralizing Antibody Assay in TZM.b1 Cells

Neutralizing antibody responses against HIV-1 Env pseudoviruses weremeasured using luciferase-based virus neutralization assays in TZM.b1cells. These assays measure the reduction in luciferase reporter geneexpression in TZM.b1 cells following a single round of virus infection.The ID₅₀ was calculated as the serum dilution that resulted in a 50%reduction in relative luminescence units compared with the virus controlwells after the subtraction of cell control relative luminescence units.Briefly, threefold serial dilutions of serum samples were performed induplicate (96-well flat-bottomed plate) in 10% DMEM growth medium (100μl per well). Virus was added to each well in a volume of 50 μl, and theplates were incubated for 1 hour at 37° C. Then TZM.b1 cells were added(1×10⁴ per well in 100 μl volume) in 10% DMEM growth medium containingdiethylaminoethyldextran (Sigma) at a final concentration of 11 μg/ml.Murine leukemia virus (MuLV) negative controls were included in allassays. HIV-1 Envelope pseudoviruses included clade A (MS208.A1 andQ23.17) isolates, clade B (SF162.LS, BaL.26, SS1196.1 and 6535.3), andclade C (MW965.26, TV1.21, ZM109F.PB4 and ZM197M.PB7) isolates.

Example 2. Generation of Optimized Mosaic Gp140 Env1 Trimers of theInvention

mEnv+(polypeptide including the amino acid sequence of SEQ ID NO: 2) hasbeen modified from mEnv (polypeptide including the amino acid sequenceof SEQ ID NO: 1) in the following manner. First, the leader peptidesecretion sequence has been made identical to that used in thestabilized clade C gp140 Env (cEnv) trimer polypeptide constituent (SEQID NO: 3). Second, cleavage site mutations have been incorporatedbetween gp120 and gp41 moieties to further enhance stability. Third, afactor Xa protease cleavage site (SRIEGR) has been incorporated upstreamof the foldon trimerization domain. The amino acid sequences of thethree Env polypeptides (SEQ ID NOs: 1-3) and the specific modificationspresent in each are depicted in FIGS. 1A-1C.

Surprisingly, these modifications resulted in a remarkably stabilizedgp140 Env1 trimer (e.g., an mEnv+ trimer of the invention). In order toassess stability, we first compared the expression levels of mEnv+relative to mEnv by Western blot analysis. To this end, T-25 flaskscontaining 80% confluent 293T cells were transfected with eukaryoticexpression vector pVRC8400 expressing mEnv or mEnv+ using lipofectamine2000 (Invitrogen) and 10 μl of each supernatant analyzed by Western blotimmunodetection using anti-Histidine tag HRP (Qiagen). FIG. 2 depicts aWestern blot showing the expression levels of mEnv and mEnv+ in lanes 3and 4, respectively. Notably, the expression levels of mEnv+ wereremarkably higher compared to that of mEnv or cEnv, which was used as apositive control (see lane 1). In this experiment, empty pVRC8400 wasused as a negative control (see lane 2).

As noted above, the mEnv+ was expressed in 293T cells and purifiedfollowing cell lysis and clarification by virtue of a His-tag using aNi-NTA (Qiagen) column. The collected fractions following imidazoleelution were pooled, concentrated, and further purified bygel-filtration chromatography on Superose 6 (GE Healthcare) in a columnrunning buffer containing 25 mM Tris (pH 7.5) and 150 mM NaCl. Achromatography trace of depicting mEnv+ elution from the Superose 6column is depicted in FIG. 3. The peak fractions (i.e., the fractionsobtained under the peak curve in FIG. 3) were then individually analyzedon a 4-15% pre-case SDS-PAGE gel (FIG. 4). The SDS-PAGE gel demonstratesthat the gel-filtration purification successfully resulted in theisolation of a homogenous population of mEnv+ polypeptides. As describedfurther herein, the immunogenicity of these stabilized gp140 Env trimers(both homotrimers of mEnv and mEnv+, as well as a combination of mEnvand cEnv homotrimers) was assessed in guinea pigs using a panel of tier1 isolates from clades A, B, and C.

Example 3. Analysis of Neutralizing Antibody Responses

Preclinical evaluation of candidate Env immunogens is critical forconcept testing and for prioritization of vaccine candidates.Luciferase-based virus neutralization assays in TZM.b1 cells (Li et al.(2005) J. Virol. 79:10108; Montefiori (2005) Curr. Prot. Immunol.Chapter 12: Unit 1211) have been developed as high throughput assay thatcan be standardized (Montefiori (2009) Methods Mol. Biol. 485:395;Polonis et al. (2008) Virology 375:315). A luciferase reporter geneassay was performed in TZM.b1 cells (a genetically engineered cell linethat expresses CD4, CXCR4 and CCRS and contains Tat-inducible Luc and(3-Gal reporter reporter genes) based on single round infection withmolecularly cloned Env-pseudotyped viruses. This assay resulted in ahigh success rate in single round infections, increased assay capacity(e.g., a two day assay), increased precision (e.g., accurately measured50% neutralization), and an improved level of standardization (e.g., astable cell line). The luciferase reporter gene assay was optimized andvalidated.

To assess the neutralization profile afforded by the stabilized gp140Env trimers of the invention, TZM.b1 assays were performed in whichguinea pig sera obtained pre-vaccination (Pre) and four weeks after thethird vaccination (Post) with cEnv homotrimers, mEnv homotrimers, orboth cEnv and mEnv homotrimers were tested against a multi-clade panelof tier 1 neutralization-sensitive isolates including clade B (SF162.LSand Ba1.26), and clade C (MW965.26 and TV1.21) HIV-1 Envelopepseudoviruses and Murine leukemia virus (MuLV) (negative control) (FIGS.5A-5C).

TZM.b1 assays were also performed in which guinea pig sera obtainedpre-vaccination (Pre) and four weeks after the third vaccination (Post)using cEnv homotrimers, mEnv homotrimers, or both cEnv and mEnvheterotrimers were tested against HIV-1 Envelope pseudoviruses ofintermediate neutralization-sensitive tier-1 (Tier 1B) clade A isolates(MS208.A1 and Q23.17) (FIGS. 6A-6B), highly neutralization sensitive(Tier 1A) and Tier 1B clade B isolates (SF162.LS, BaL.26, SS1196.1, and6535.3) (FIGS. 7A-7D), and Tier 1A and Tier 1B clade C isolates(MW965.26, TV1.21, ZM109F.PB4, and ZM197M.PB7) (FIGS. 8A-8D).

Unexpectedly, quantitation of ID₅₀ titer data collectively demonstratethat the combination of cEnv and mEnv homotrimers induced neutralizingantibody responses that were superior to either cEnv or mEnv alone.Specifically, the combination of cEnv and mEnv was particularlysurprising in terms of expanding the breadth of neutralizing antibodyresponses induced. Such an expansion of neutralizing antibody breadthhas not previously been described and is a major unmet need in thefield.

Example 4. Treating or Reducing the Risk of an HIV Infection in aSubject Using the Compositions of the Invention

The composition of the invention (e.g., a vaccine of the invention) maybe administered to a subject (e.g., a human infected with HIV or at riskof an HIV infection) in a prime-boost vaccination regimen to treat orreduce the risk of an HIV infection in a subject in need thereof. Forexample, one or more of the compositions of the invention, such asvaccine including mEnv, mEnv+, or cEnv trimers, or combination of mEnvor mEnv+ and cEnv trimers may be administered as a boost. Prior toadministration of the boost, the subject is administered as a primevaccination at least a first vector including a first nucleic acidmolecule that encodes a polypeptide having at least 85% amino acidsequence identity (e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identity) to, or the sequence of, SEQ ID NO:6, and optionally a second vector including a second nucleic acidmolecule that encodes a polypeptide having at least 85% identity (e.g.,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identity) to, or the sequence of, SEQ ID NO: 7.

The composition is preferably administered in an amount that provides asufficient level of the stabilized gp140 Env trimer gene product (e.g.,a level of stabilized gp140 Env trimer that elicits an immune responsewithout undue adverse physiological effects in the subject caused by theimmunogenic trimer). If the composition is non-vectored, the polypeptidecomposition administered may include between approximately 1 μg and 1 mgof stabilized Env trimers, and more preferably between 50 μg and 300 μgof stabilized Env trimers of the invention. Alternatively, the subjectmay be administered, in the form of a viral vector, at least about 1×10³viral particles (vp)/dose or between 1×10¹ and 1×10¹⁴ vp/dose,preferably between 1×10³ and 1×10¹² vp/dose, and more preferably between1×10⁵ and 1×10¹¹ vp/dose.

Following administration of the composition of the invention in aprime-boost regimen, the patient can be assessed for changes in one ormore symptoms or, in particular, the level of HIV titer in the treatedsubject, and the regimen can be repeated as necessary as describedherein above.

OTHER EMBODIMENTS

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure that come within known or customary practice withinthe art to which the invention pertains and may be applied to theessential features hereinbefore set forth.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindependent publication or patent application was specifically andindividually indicated to be incorporated by reference in theirentirety.

What is claimed is: 1-37. (canceled)
 38. A protein comprising aminoacids 30-724 of SEQ ID NO:2.
 39. A pharmaceutical composition comprisingthe protein of claim 38 and a pharmaceutically acceptable carrier,excipient, or diluent.
 40. The pharmaceutical composition of claim 39,further comprising an adjuvant.
 41. The pharmaceutical composition ofclaim 40, wherein the adjuvant comprises an aluminum salt.
 42. Thepharmaceutical composition of claim 41, wherein the adjuvant comprisesaluminum phosphate.
 43. The pharmaceutical composition of claim 39,further comprising one or more additional HIV gp140 polypeptides. 44.The pharmaceutical composition of claim 43, wherein the one or moreadditional HIV gp140 polypeptides comprise at least one polypeptidecomprising amino acids 30-708 of SEQ ID NO:3.
 45. A compositioncomprising: (i) a first protein that comprises three gp140 polypeptides,wherein each of said three gp140 polypeptides comprises an amino acidsequence comprising amino acids 30-724 of SEQ ID NO: 2; (ii) a secondprotein that comprises three gp140 polypeptides, wherein each of saidthree gp140 polypeptides comprises an amino acid sequence comprisingamino acids 30-708 of SEQ ID NO: 3; and (iii) aluminum phosphate. 46.The composition of claim 45, being a pharmaceutical composition.
 47. Thecomposition of claim 45, wherein the first protein and the secondprotein both are oligomeric proteins.
 48. A method of preparing acomposition, the composition comprising: (i) a first protein thatcomprises three gp140 polypeptides, wherein each of said three gp140polypeptides comprises an amino acid sequence comprising amino acids30-724 of SEQ ID NO: 2; and (ii) a second protein that comprises threegp140 polypeptides, wherein each of said three gp140 polypeptidescomprises an amino acid sequence comprising amino acids 30-708 of SEQ IDNO: 3, the method comprising providing the first protein and the secondprotein and mixing the first and second protein into a composition. 49.The method of claim 48, further comprising providing an adjuvant andmixing the adjuvant into the composition.
 50. The method of claim 49,wherein the adjuvant comprises an aluminum salt, such as aluminumphosphate.
 51. A method of inducing an immune response against HIV in asubject, the method comprising administering to the subject thepharmaceutical composition of claim
 39. 52. A method of inducing animmune response against HIV in a subject, the method comprisingadministering to the subject the pharmaceutical composition of claim 46.53. A method of inducing an immune response against HIV in a subject,the method comprising administering to the subject a composition thathas been obtained according to claim
 48. 54. The method of claim 51,wherein the pharmaceutical composition is administered intramuscularly,intradermally, subcutaneously, or mucosally.
 55. The method of claim 51,wherein said subject is administered at least one dose of saidcomposition.
 56. The method of claim 51, wherein the pharmaceuticalcomposition is administered to said subject as a prime, a boost, or as aprime-boost.
 57. The method of claim 52, comprising intramuscularadministration.